TM/SC outflow enhancing drugs are also effective ocular hypotensive agents . One of the most effective anterior chamber AQH microshunt devices is the Preserflo® microshunt which can lower IOP down to 10–13 mmHg. Other IOP-lowering drugs and devices on the horizon will be also discussed. Additionally, since elevated IOP is only one of many risk factors for development of glaucomatous optic neuropathy, a treatise of the role of inflammatory neurodegeneration of the optic nerve and retinal ganglion cells and appropriate neuroprotective strategies to mitigate this disease will also be reviewed and discussed.Damage to the optic nerve and the death of associated retinal ganglion cells (RGCs) by elevated intraocular pressure (IOP), also known as glaucoma, is responsible for visual impairment and blindness in millions of people worldwide. The ocular hypertension (OHT) and the deleterious mechanical forces it exerts at the back of the eye, at the level of the optic nerve head/optic disc and lamina cribosa, is the only modifiable risk factor associated with glaucoma that can be treated. The elevated IOP occurs due to the inability of accumulated aqueous humor (AQH) to egress from the anterior chamber of the eye due to occlusion of the major outflow pathway, the trabecular meshwork (TM) and Schlemm’s canal (SC). Several different classes of pharmaceutical agents, surgical techniques and implantable devices have been developed to lower and control IOP. First-line drugs to promote AQH outflow via the uveoscleral outflow pathway include FP-receptor prostaglandin (PG) agonists  and a novel non-PG EP2-receptor agonist (omidenepag isopropyl, Eybelis Human and animals heavily rely on good vision to perform their daily tasks and for survival, and indeed eyesight is undoubtedly the most valuable of our precious senses. The eyes being windows for the brain is apt since 80% of the external information reaching the neural networks comes in from the visual system. Thus, visual impairment in any form has a devastatingly negative impact on most people whose greatest fear is blindness. Sadly, the World Health Organization  reports Before delving into the pathological basis of glaucoma, it is necessary to describe the anatomical structures and the functions of the visual axis. The mammalian eye is exposed to the outside world somewhat unprotected although the eye socket offers some protection. Similarly, the protective thick white fibrous outer layer (sclera) of the eyeball affords the eye its shape while insulating the interior components from possible damage. The transparent cornea at the front of the eye consists of five layers of different cell types and represents a specialized scleral tissue . The conMYOC, MGP and PDPN), SC cells and resident macrophages. However, the structural elements of the TM were composed of Schwalbe’s line cells  in the non-filtering area of the TM around the juxtacanicular region. The juxtacanicular cells differentially expressed various genes including CH13L1, ANGPTL7, RSPO4, FMOD and NELL2. Interestingly, SC cells displayed a profile made up of both lymphatic endothelia and blood macrophages. Lastly, there were differences in the genetic expression profiles of TM cell-types  and SC cells , with differences extending to genes involving OHT-related high IOP vs controls. Differences were also observed in RGCs genes which were unrelated to elevated IOP. Therefore, the collective studies from several groups highlight the fact IOP generation and regulation are mediated by a complex array of cell-types in the outflow pathways, and that these elements appear to be well conserved across multiple species The AQH also nourishes the cells lining the ANC of the eye as it percolates through the ANC to exit via the angular space between cornea and iris via a filtration system, trabecular meshwork (TM), that is connected to the Schlemm’s canal (SC) that allows the AQH to pass into the veinous circulation via series of complex plexi of smaller vessels ,B, 2A,B.Additional single-cell transcriptomic investigations of human ANC cells revealed up to twelve distinct cell-types . Of the In contrast, the posterior segment of the eye behind the lens is filled with a jelly-like material (vitreous humor [VH]), and this chamber is substantially larger than the ANC. The VH, composed of many different proteins and water, also helps in shaping the eyeball and also provides much cushioning and protection to the retina that lines the inside of sclera at the back of the eyeball, and it probably is also involved in removing some of the waste products of the retina. Just as the cornea at the front of the eye is composed of many cell layers, the retina is highly complex and contains many specialized cell-types.Since glaucoma results from structural and functional failures at multiple levels throughout the visual system, it is important to be aware of the composition and organization of the various components. In the rear of the human eyeball, the retina contains many different types of cells that are also essentially transparent such that light reaches the back of the eye and penetrates to the deeper layers of the retina where the photo-sensitive cells  reside. Whilst cones are concentrated in a central region of the retina called fovea, the rods are mostly located in the peripheral regions of the retina. Whereas the cones are specialized for high acuity tasks like reading and color perception, rods are responsible for night vision and respond best to dim light .The jelly-like VH fills the majority of the posterior segment of the eye. At the back of the eye, the VH is separated from the retinal tissue by the inner limiting membrane. Next comes a multilayered retinal nerve fiber layer (RNFL) composed of the axons of the RGCs that converge at the optic nerve head (ONH)/optic disc area , pass thThe retina is a high energy-demand tissue and it receives nutrients and oxygen from the retinal arteries and the choroid . The bloThe dura, arachnoid and the pial membrane sheaths encase the optic nerve  which isMuch joy and happiness comes to us through our eyes as we appreciate the beauty around us, especially the display mother nature arranges for us. It is no wonder then that eyesight is so precious.+-channels are closed to allow intracellular levels of Na+ ions to build-up and the cell membrane potential becomes positive. Now, the Ca2+-channels are closed, and inhibitory transmitter levels are decreased which removes the inhibition and the bipolar cells are stimulated/excited creating an actional potential in the RGCs which are sent down their axons to the thalamic brain nuclei. Defects in the membrane repolarization process and locally produced endothelin impairs axonal transport and deprives RGCs of neurotrophin support from the brain  of the thalamus . The optAs described above, most optic nerve projections from the retina traverse to the LGN which sorts retinal signals into parallel streams containing color, structure, motion and contrast. Since the LGN is also structurally layered, its top four parvocellular layers handle color and fine structure, while the bottom two magnocellular layers process contrast and motion. Not surprisingly, the primary visual cortex is also highly organized to receive LGN inputs to maintain fidelity and complexity of the information being sent to it. The LGN magnocellular and parvocellular layer cells send long axons to the ancient part of the brain comprised of the primary visual cortex (PVC) . Here inFocusing on the ANC of the eye will now permit a detailed description and discussion about ocular hypertension (OHT) and glaucoma. This ocular disease is represented by a group of pathological conditions which all culminate in the death of RGCs and eventual loss of much of their axonal connections to the brain structures mentioned above . The disMany different forms of glaucoma are known . The mosSecondary glaucomas, as the name implies, occurs due to an ocular insult or due to another medical problem such as ANC inflammation (causing uveitic glaucoma) or due to steroid-induced accumulation of complexed extracellular matrix proteins in the TM that prevent AQH efflux, for example. Neovascular glaucoma occurs when abnormal growth of new blood vessels obstructs the AQH drainage pathways in the ANC and it is usually caused by diabetes or high blood pressure. Pigment dispersion syndrome (pigmentary glaucoma) results from aberrantly released melanin granules from the iris which then occlude the AQH drainage pathways thereby causing OHT. Myopic young white men are prone to pigment dispersion syndrome than other people. The most well known animal model of pigmentary glaucoma is the DBA/2J mouse and its variants . Lastly,rd largest cause of blindness worldwide, and glaucoma associated with uveitis is a very serious disease requiring immediate attention since the IOP can rise suddenly and to a high magnitude , low intracranial fluid pressure, low retinal perfusion, advanced age, African-Caribbean-Latin American ancestry, family history of glaucoma, thin corneas, myopia, diabetes, high blood pressure and low diastolic pressure. PACG occurs suddenly when the iris is displaced and blocks the AQH drainage pathway causing a rapid elevation of IOP, perhaps as high as 70 mmHg. Its clinical manifestations include nausea, blurred vision, ocular pain, cloudy corneas and halos around lights. The risk factors linked to PACG include: advanced age, shallow ANC angles, Asian-Eskimo ancestry, family history of PACG, hyperopia and female gender.Several clinical trials conducted in the late 1900s early 2000s revealed that lowering of the IOP is highly beneficial and directly slows down and can prevent glaucoma progression in most forms of glaucoma . Consequ2, glucose and growth factors) and a stable environment  for the cells/tissue lining the ANC  of the CP within the CB generate the clear AQH fluid by passive diffusion, ultrafiltration and secretion, with the latter being the predominant event  synthase and release the vasodilator gaseous transmitter NO under pressure to compensate for the increased resistance  which represents ∼75% of all forms of glaucoma. Primary closed-angle glaucoma (PACG) has the second highest occurrence (∼23 million patients). Unfortunately, Asian and African nations have the highest number of POAG and PCG patients, greatly outnumbering those afflicted with these glaucoma forms in Europe and North America. Close to 5 million patients are projected to have POAG in North America by 2040.Akin to most chronic diseases, POAG has a age-related occurrence with patients being diagnosed with this disease almost exponentially between age 40 to age 80 in almost all geographical locations. POAG is asymptomatic, the patient feels no pain and is oblivious to the disease development since other warning signs are all absent. This “silent thief of sight” makes its detection and diagnosis difficult, and it is estimated that 50% of future POAG patients remain undiagnosed. Additionally, during early stages of POAG/PCG/NTG, the patient does not notice much change in the vision and the brain compensates  for loss2 species . Historically, bacterial and viral infections led to the development of the mammalian immune system . SubsequThe classic debate of which happens first, failed function or failed structure is a complex one. Regardless, structural changes within the optic nerve and retina precede the functional visual deficits resulting from chronic OHT, POAG/PACG and NTG. Temporally, the lag period between onset of OHT and glaucoma diagnosis contributes heavily to visual deficits, and thus preservation of vision requires the earliest possible diagnosis of these asymptomatic diseases. The strong correlation between structural damage throughout the visual system and eyesight deficits of animals and humans with glaucoma has been confirmed by numerous investigators . SimilarOne intriguing possibility that has some experimental support is that GON has an autoimmunogenic component in addition to the IOP-induced component . Thus, sSeveral pioneering clinical trials in the early 2000s and animal models of OHT/glaucoma have provided compelling evidence that elevated IOP/OHT is a principle root cause of several forms of glaucoma. Consequently, a multipronged approach has been adopted to address this aspect of the disease. Thus, reducing IOP with ocular hypotensive drugs, removal of excess AQH from the ANC of the eye with microshunts and/or surgically promoting AQH drainage from the ANC of the eye of OHT/POAG/NTG patients slows down disease progression and preserve sight by reducing the death of RGCs and their axons , carbachol (1932), acecledine (1960), propanolol (1967), and clonidine (1972). However, many of these drugs were relatively non-selective for the family of receptors through which they imparted their ocular hypotensive activity. While more potent and slightly longer acting beta-adrenoceptor antagonists were soon discovered and approved for glaucoma treatment , it was only thereafter that receptor selectivity began to be addressed. Thus, betaxolol  administered therapeutics utilized to lower and maintain IOP include several FP-receptor prostaglandin (PG) agonists such as latanoprost, travoprost, tafluprost and bimatoprost which all enhance UVSC outflow of AQH in POAG/NTG patients and are used once-daily  Sharif . Recent TM outflow promoting drugs include the muscarinic receptor agonist pilocarpine , rho kinLast but not least, there are pharmaceutical agents that slow down the production of AQH in order to decrease IOP see . Such AQEven though the health authority-approved ocular hypotensive drugs described above are effective at reducing IOP they all have various types of ocular, and in some cases, systemic side-effects . Notably+-channel activators  utilized to insert the device and the type of microshunt that is utilized to drain the AQH from the ANC to lower IOP. Additionally, the target patient population, whether a bleb is used, the overall IOP-reduction achieved, ocular advere-events observed, and the relative need for t. o. drugs post-operation to maintain IOP help differentiate between the various MIGS available today. Since only modest reductions in IOP have been achieved with most MIGS procedures thus far, there remained an unmet medical need for a MIGS treatment to deal with moderate-to-severe and refractory glaucoma (® MicroShunt (formerly termed the InnFocus MicroShunt)  . This Microshunt –14. Thiscroshunt  , including testing in receptor subtype-based/enzyme isoform-based assays in order to determine relative selectivity of the compounds for the desired target protein (sub-type or isoform). Thus for example, for 5-hydroxy tryptamine-2  receptor agonist IOP-lowering compounds, not only was the affinity, agonist potency and intrinsic activity required at each of 5-HT2 receptor sub-types  but also at each of the 6 other major 5-HT receptor classes , and if possible at their subtypes  mobilization  is the identification of a suitable target to pursue that addresses an unmet medical need as described or defined by key opinion leaders, clinicians and patients of the OHT/POAG field. The target concept profile (TCP) or target product profile (TPP) then needs to be defined based on clear, objective, achieveable and desired attributes of the final drug candidate. Some of this information may exist in the literature or may be entirely proprietary. However, the knowledge of the target protein and disease mechanism and/or pathway is important in order to render the target into a compound screening funnel. In order to ensure that the target mRNA/protein is located in the normal and diseased target cells/tissues needs to be ascertained using RT-PCR , or in sdization , or recedization  and/or idization  techniquls e.g.,  should bcampaign . The inig., cAMP /cGMP ; inositg., cAMP /intracellization   can also be ascertained by sampling the effluent from these perfused segments. Such data has been reported using partially intact ex-vivo ANC eye segments of numerous different species , excessive blinking, tearing and conjunctival discharge, swelling of the eye lids, and vocalization in single or multi-dose topical ocular studies. The vehicle alone should be used as a control in the contralateral eye. When compounds pass the acute/chronic ocular safety process, and general systemic/central nervous system (CNS) safety , they can then be tested for their ability to lower IOP. If test agents are suspected of having a topical ocular anesthetic effect, they can be tested for such activity, and/or irritability potential, using electrophysiological recordings of corneal nerves but often these tests are performed after reproducible IOP-lowering effects have been determined e.g., .Following successful passing through the ocular irritation stage-gate, compounds can now be tested for their ability to lower IOP. The test agents are again prepared in the selected formulation and are dosed t. o. at a single or multiple concentrations/doses to determine their relative efficacies to reduce IOP. Since the non-human primates are the most precious, costly and more labor-intensive to train and utilize for ocular investigations, rodents, guinea pigs and rabbits are preferred in early-stage efficacy studies. Testing for ocular hypotensive activity is usually performed in normal eyes with normal physiological IOPs. However, in order to mimic the human OHT/glaucoma condition, animals with experimentally or genetically induced  OHT are OHT eyes of rodents and rabbits can be created using a variety of techniques using hypertonic saline injection into the episcleral veins , photocoagulation of these veins , injectiIf a high capacity screening model is desired for testing a large number of potential IOP-lowering agents then rodents are probably the best animal model. They are relatively cheap, easier to handle, cheaper to maintain and breed. The similarity of the mouse eye structure and genomics to humans are additional benefits . Furtherex-vivo models lend themselves to further mechanistic investigations since various enzyme inhibitors, receptor antagonists or other drugs can be perfused and their effects studied on the outflow parameters. Some examples of compounds tested recently in such ex-vivo models that stimulated outflow include a non-peptide bradykinin-mimetic (FR-190997), and a mixed dopaminergic and 5HT2 receptor agonist (cabergoline)  . One stutagonist .+) or its congeners, and/or abnormal mitochondrial metabolism/catabolism within retinal neurons of patients with NTG/POAG may heavily contribute to their GON and visual deficits , they continue to experience vision loss. Also, it is estimated that 30–80% of OHT/glaucoma patients’ disease continues to progress despite adequate IOP reduction. Conversely, many humans have abnormally elevated IOPs but they do not progress to GON and their vision is unaffected by the raised IOP. These observations strongly suggest that either IOP-independent events and pathologies underlie the development of GON in the NTG patients or that their visual system components are much more sensitive to the IOP levels which are considered “normal”. The same may apply to other patients with other forms of glaucoma. Indeed, accumulating evidence indicates that OHT alone is not the sole contributor to glaucomatous damage and the resultant visual impairment as discussed earlier. OHT and other damaging events ongoing simultaneously probably conspire to cause visual impairment in NTG/POAG/PACG patients. Thus, IOP-independent mechanisms involving retinal vascular dysfunctions  and the deficits . Hence, in vitro assay systems using a variety of animal and human isolated retinal, LC and ONH cells subjected to various chemical and mechanical stressors . Delivery of mitochondrial-targeted anti-oxidants (Significant progress has been made in the discovery and characterization of preclinically effective neuroprotectants/cytoprotectants at least from sors see . The bursors see , B-vitamsors see , antagonsors see , valproisors see , Ca2+-chdulators , ATP-senompounds , neurostompounds , α2-adreompounds , mTOR inompounds , etc., eoxidants , inactivoxidants , use of oxidants , RGC andoxidants , migrogloxidants , deliveroxidants , and diroxidants . Directloxidants  may suppoxidants .Of the drugs that have been clinically evaluated for neuroprotective activity and to retard vision loss or impairment in POAG patients, for example brimonidine and memantine, none have conclusively proven effective thus far . It is win vivo models of GON as described above (The above discourse provides strong evidence for involvement of defective anterior chamber physiology in the onset of POAG, PACG and other forms of glaucoma. Specifically, the aging process leads to energy depletion in mitochondria  and celled above . Howevered above  and DARCed above , adaptived above , angiogred above , and of ed above  should aed above . Perhapsed above , regenraed above , also hoed above  will offed above .


Recent advances have unraveled the complexity of molecular signals that allow the communication between different reproductive cell types for maintaining normal reproductive functions. This kind of communication is controlled by a myriad of factors acting through autocrine, paracrine, and endocrine signaling pathways . In the Qamar et al. reviewed the active participation of EVs with the male and female reproductive systems in the regulation of different physiological events such as oocyte maturation, fertilization, and embryo and fetal development. To illustrate the EVs functions in the ovary, Uzbekova et al. profiled the proteins within bovine follicles EVs of 53.6 ± 23.3 nm in diameter and identified 322 unique proteins, which originated from granulosa cells and from other cells. These proteins may be involved in the maintenance of follicular homeostasis and may affect oocyte developmental competence. Moreover, Mazzarella et al. investigated the micro RNAs cargo of oviduct fluid small EVs (sEVs) of around 140 nm in size in pregnant cows. They found that some unique miRNAs  were up-regulated in pregnant cows at 120h after ovulation induction and one miRNA (bta-miR-331-5p) was up-regulated in non-pregnant cows. The results suggest that initial embryonic-maternal communication is mediated through the EVs in the oviduct, and the presence of gametes and the embryo can modulate miRNAs contents of oviduct EVs. Regarding the male-related factors, Xu et al. examined the RNA contents of boar seminal plasma EVs by NGS and identified many RNA classes, including mRNA (25%), PIWI interacting RNA (15%), and miRNA (9%). A total of 288 miRNAs with 37 novel miRNAs, and 19,749 piRNAs, were identified in the boar seminal plasma EVs. Particularly, ssc-miR-21-5p was identified, which may confer negative effects on boar sperm fertility based on a dual-luciferase reporter experiment.Recognized researchers in this field worldwide have been invited to contribute to the current Research Topic. We received three original articles and three reviews discussing the current advances in EVs isolation and characterization for a better understanding of sperm physiology, ovarian follicles function, and oviduct secretions during pregnancy in different animal models. Profiling the protein and RNA cargo in these EVs provides a powerful diagnostic tool for accurate and non-invasive assessment of male and female reproductive functions. Moreover, the EVs contents exert possible pleiotropic effects on both male and female reproductive tissues and germ cells, as well as on embryo early development, implantation, and further fetal growth. Talebjedi et al. reviewed the conventional methods for isolating EVs and compared them with the most advanced microfluidics methods. Moreover, they discussed the role of embryonic EVs as biomarkers of embryo quality which could be implemented in the future as a non-invasive diagnostic test for embryo selection. Then they showed the promising potentials in which microfluidics technologies could allow researchers to overcome the challenges of embryonic EVs isolation and be used as a fast and user-friendly tool. Of note, Gebremedhn et al. summarized the potential roles of EVs in mediating the follicular cells communication and shuttle the effects of environmental and metabolic stressors in various reproductive cells. Interestingly, EVs are involved in shuttling protective or rescuing molecular signals during oxidative, heat, and metabolic stress. Their future potential usage as means of targeted delivery of molecules to alleviate the effect of different stressors on oocytes was also discussed.Challenges still remain in isolating EVs from the minute embryo culture medium quantity with low EVs concentration, and much effort is required to resolve the use of these EVs as biomarkers for non-invasive determination of embryo developmental potential. For that, All together, these papers show that a better understanding of the molecular mechanisms mediating the embryonic-maternal crosstalk through EVs will lead to the development of new regulating agents , as wellAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Despite their small size, the membrane-bound particles named extracellular vesicles (EVs) seem to play an enormous role in the pathogenesis of acute leukemia. From oncogenic hematopoietic stem cells (HSCs) to become leukemic cells to alter the architecture of bone marrow (BM) microenvironment, EVs are critical components of leukemia development. As a carrier of essential molecules, especially a group of small non-coding RNAs known as miRNA, recently, EVs have attracted tremendous attention as a prognostic factor. Given the importance of miRNAs in the early stages of leukemogenesis and also their critical parts in the development of drug-resistant phenotype, it seems that the importance of EVs in the development of leukemia is more than what is expected. To be familiar with the clinical value of leukemia-derived EVs, this review aimed to briefly shed light on the biology of EVs and to discuss the role of EV-derived miRNAs in the development of acute myeloid leukemia and acute lymphoblastic leukemia. By elaborating the advances and challenges concerning the isolation of EVs, we discuss whether EVs could have a prognostic value in the clinical setting for leukemia. As the most common type of leukemia, the incidence of acute leukemia, either acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL), is increased in the last few years . The aggRecently, a group of extracellular vesicles (EVs) derived from the cancer cells that carry the property of their parental cells ranging from signaling proteins to nucleic acids such as DNAs was successfully identified . It becaIn addition to their key roles in the pathogenesis of acute leukemia, EVs are also a valid repertoire of genetic and epigenetic abnormalities of the parental cells. The next-generation sequencing (NGS) and GeneScan-based fragment-length analysis on the identified double-stranded DNA (dsDNA) in AML-derived EVs revealed that the majority of dsDNA mirror the mutations found in the genomic DNA obtained from primary leukemia cells . It has The evidence of the existence of EVs dated back to almost 60 years ago when the 20–50 nm-sized vesicles carrying clotting factors were identified in human platelet-free plasma . In an aLike their intracellular functions, the biogenesis of circulating vesicles is complex and numerous internal and external factors are involved in this process. Different membrane receptors, lipid raft complexes, and endosomal sorting complexes regulate the biogenesis of EVs; however, this process could not be accomplished without the presence of Ras superfamily GTPase .The biogenesis of exosomes includes three main steps; early endosomes (EEs) and late endosomes (LEs) or MVB formation, intraluminal vesicles (ILVs) formation, and the secretion of ILV into the extracellular space.Early endosomes (EEs) form as a result of membrane budding inward the cell. The generated EEs subsequently fuse with endocytic vesicles to incorporate their cargos. After the evacuation of recycling endosomes from EEs, they undergo several stages of maturation to produce late endosomes (LEs), also known as multivesicular bodies (MVBs). There are two destinies for MVBs; either merge with the lysosome which leads to their destruction, or package their contents as 30–100 nm vesicles named intraluminal vesicles (ILVs) to integrate with the plasma membrane and release into the extracellular space . In thisThe formation of ILVs consists of two main steps. At the primary stage, the endosome membrane should be organized by specific transmembrane proteins known as Tetraspanins . The previa sphingomyelinase 2 produces ceramide, a waxy lipid molecule which facilitates the formation of ILVs through regulating membrane budding (via interaction with inhibitory G protein (Gi)-coupled receptor. No matter through which mechanism ILVs may be produced within the MVBs. NSF attachment protein receptors (SNAREs) provide a platform for their secretion into the extracellular space. Through binding of Ca2+ to synaptotagmin VII, SNAREs complex could be activated, resulted in secretion of EVs  differ from the size from the exosomes, their biogenesis might have some differences with EVs. In the biogenesis of MV, actin rearrangement play a fundamental role . In GTPaBased on the type of the parental cells and the mission that EV may have, the cargo of these circulating vesicles varies. However, proteins, lipids, metabolites, RNAs, and cDNAs are the common components of EVs . Table 1Apart from structural proteins such as tetraspanins , TSG101, Alix, and Rab family, heat shock protein, clathrin, protein kinase G (PKG), ATPase, syntenin, and RNA binding proteins (RBPs) are the most common types of proteins that are detected in EVs . It shouOne of the most important contents of EVs is nucleic acids that could be either mitochondrial DNA (mtDNA), double strands DNA (dsDNA), single strands DNA (ssDNA) , or diffvia inducing chromatin remodeling at enhancer site  or membrane proteins, miRNAs could be packaged into EVs . For exavia either endocytosis or membrane fusion. Once EVs enter the cells, they evacuate their valuable biologically active molecules repertoire to alter the cellular behavior. For example, the delivered miRNAs or lncRNAs could epigenetically change the expression of a wide range of molecules, activate/suppress different signaling pathways, and change the chromosomal structure. Also, the transferred mRNA could be translated into proteins that previously did not exist in the responder cells , for example, AML-derived EVs could halt the progression of hematopoiesis and osteoblast differentiation in the BM niche and promote the uncontrolled proliferation of leukemic cells . BMP-2 iSince the density of BM microvessels is one of the main criteria that could protect the survival of leukemic cells in the BM, it comes as no surprise that EVs might participate in the regulation of the density of BM microvesicles. Through delivering angiogenic factors/proteins and miRNAs, EVs change the characteristics of endothelial cells and promote angiogenesis . The angConstructing a leukemia-friendly environment, EVs also protect leukemic cells from anti-cancer agents. The number of studies demonstrated the role of exo-miRNAs/proteins in conferring drug resistant phenotype is rare in ALL cases. It has been reported that when BMSCs absorb leukemia-derived EVs, they would be transformed into cancer associated fibroblasts (CAFs) that could evolve a protective niche against chemotherapeutic drugs . On the Given the importance of EVs in the pathogenesis of acute leukemia, many studies have come to a consensus that analyzing the contents of these circulating vesicles could provide a valuable perspective about the outcome of patient with leukemia. Given the importance of EVs in the pathogenesis of leukemia and based on the number of reports suggesting the benefits of evaluating EVs contents for early diagnosis or predicting the outcome of patients, it could be known that EVs are promising circulating biomarkers. However, the story is not as simple as it looks. The study of EVs as biomarkers in clinical approaches is still a new field, and no standard methods have been established yet for the proper enrichment and isolation of these circulating vesicles. The diversity in the protocols used for EVs isolation, enrichment, and measurement leads to the fact that in many cases, the results of studies are not comparable with each other, and this may have a negative effect on the validation of the results . For exaThe diversity in the isolation techniques could not only produce variability in the results but also influence the precise molecular characterization of EVs. The high purity of EVs and ability to distinct the EV-derived proteins/nucleic acids from non-EV sources are essential for the proper characterization of EVs. Apart from this, thus far, several techniques, including transmission electron microscopy (TEM), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), flow cytometry, and tunable resistive pulse sensing (TRPS) are used for the physical characterization of EVs. Although effective, each of these techniques also have some limitations and complications. Problems such as vesicle shrinkage and the limited number of vesicles that could be analyzed by TEM have made flow cytometry superior to TEM which is currently a gold standard method . Howeverin vitro analysis. So, experiments conducting on patient’s samples are required to achieve better results and provide a wider perspective about the application of EVs as a prognostic factor in patients with acute leukemia.The application of EVs in risk stratification for patients with acute leukemia is beneficial, though it has a long way to be achievable. First, as mentioned earlier, due to the limitation of all mentioned isolation techniques in distinguishing exosomes from micro-vesicles, both of these cellular components referred to as circulating vesicles, which limits our understanding of the property of these circular vesicles. The less we know about the contents of circulating EVs, the longer it might take for receiving approval EVs as a diagnostic tool in clinical application . MoreoveFrom the first description of EVs in the samples of patients with leukemia, there is no doubt that these tiny circulating vesicles play fundamental roles in leukemogenesis, cell proliferation, survival, and also angiogenesis. The components of EVs conveniently alter the structure of BM in the way that it protects leukemic cells from either the adaptive arm of the immune system or anti-cancer agents. Thus far, many studies are focusing on the importance and value of EVs in determining the outcome of patients with leukemia; however, a long way should be passed to reach the best results. As it was mentioned earlier, according to the size of EVs, three classes of these circulating vesicles have been identified in body fluids and for sure, each of them may participate in some specific biological processes. However, due to the size overlap and the disability of the current isolation and characterization technologies, it is impossible to distinguish these vesicles from each other and analyze their cargos individually. Moreover, many of the results published in this area are conflicting and incomparable, as each research group might use different techniques for the collection, processing, and storage of EVs. Given these limitations, it seems that more issues should be addressed before EVs could enter into the clinical application for acute leukemia. Nevertheless, the journey of EVs in leukemia is still mesmerizing.MI: conceptualization, literature survey, figure designing, review structure, writing review and editing, and references collection. ZH: conceptualization, literature survey, review structure, writing review and editing, and references collection. FJ: literature survey, writing review, and references collection. AH and AG: critical review and editing. Y-DL: formating and reference collection. LJ: conceptualization, literature survey, writing review and editing, references collection, tables preparation, supervision, and correspondence. Z-SC: critical review and editing, correspondence, and supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Exosomes from extracellular vesicles can activate or inhibit various signaling pathways by transporting proteins, lipids, nucleic acids and other substances to recipient cells. In addition, exosomes are considered to be involved in the development and progression of tumors from different tissue sources in numerous ways, including remodeling of the tumor microenvironment, promoting angiogenesis, metastasis, and invasion, and regulating the immune escape of tumor cells. However, the precise molecular mechanisms by which exosomes participate in these different processes remains unclear. In this review, we describe the research progress of tumor cell-derived exosomes in cancer progression. We also discuss the prospects of the application of exosomes combined with nanoengineered chemotherapeutic drugs in the treatment of cancer. Extracellular vesicles (EVs) are membrane-bound, nanosized vesicles that are released from different cell-types and are able to transport nucleic acids, proteins, and other cellular cargo . EVs comIntercellular communication is extremely important for various cells to adapt to changes in the different intracellular and extracellular environments and can occur at different processes and stages, such as during embryonic development, in response to trauma, and in the maintenance of homeostasis in the organism . The mecDuring the maturation of multivesicular endosomes (MVES), exosomes are considered to be intraluminal vesicles (ILVs), which are formed via inward budding of the endosome membrane. They are intermediates in the endosome system and are secreted when MVES fuse with the cell surface , 14. ExoStudies on exosomes and their biological functions have improved our understanding of the intercellular communication of exosomes in different cell types. These nanoscale vesicles are effective carriers of the regulatory information of biological macromolecules, and can be further induced and regulated by the receptor cells. The main cell types including fibroblasts, endothelial cells, and immune cells that interact with cancer cells through exosome signaling in the tumor microenvironment. The outcome of the interactions mentioned above relies on the origin of the exosomes and their exosomal cargo , 17. HypProliferation of tumor cells is an essential process for cancer progression, and this process is dependent upon growth factors. Growth factors can also support the tumor microenvironment . By releIn addition, tumor cell-derived exosomes can modify the migratory status of recipient malignant cells . These eExosomes are considered to be involved in the progression of various precancerous liver diseases, including viral hepatitis, alcoholic liver disease, and even the progression of liver fibrosis, which eventually develops into hepatocellular carcinoma (HCC) . For exade novo “off the shelf” metabolites through exosomal cargo (Exosomes derived from cancer-associated fibroblasts (CAFs) have been shown to provide certain nutrients to pancreatic and prostate cancer cells and to drive them to glycolysis. Hongyun Zhao et al. demonstrated that CAFs-derived exosomes (CDEs) from patients reprogram cancer cell metabolism by disabling mitochondrial oxidative metabolism and providing al cargo . Xiaofenal cargo . Examinaal cargo . On the al cargo . Additioal cargo Figure 1In situ tumor cells can also undergo this change, which gives them the ability to invade and metastasize , can stimulate glioblastoma movement, invasion, and proliferation, resulting in a poorer prognosis for patients with glioma . ExosomaIn conclusion, the above studies suggest that tumor cell-derived exosomes regulate the metastasis and migration of tumor cells through different types of proteins and miRNAs Table 1.The activity of exosomes affects five major functions of immune regulatory mechanisms: immune activation, antigen expression, immune surveillance, immunosuppression, and communication between immune cells. In addition to immune cells, tumor cells can also secrete immunoreactive exosomes, thus influencing pathophysiological processes –67. Furt+ exosomes carried HCV particles and established an environment for persistent HCV infection to promote the progression of liver cancer through immune escape  could promote CTL generation, while Treg-cell-derived exosomes could inhibit CTL generation, and that exosomes from immune cells and tumor cells could regulate cancer progression . Tumor ce escape .In addition to exosomes regulating tumor immune escape, proteins and nucleic acids contained in exosomes can also be used as substances detected by immune checkpoint blockade technology. Due to previous technical limitations, exosome extraction has often been difficult. However, with the development of microfluidic technology, RNA sequencing technologies, and the rapid development of proteomics, the use of exosomes as a diagnostic and prognostic biomarker for immune checkpoint blockade technology has become possible .As a treatment option, exosomes are characterized by the lack of toxic side effects and rejection, and can therefore be used as a vehicle for drug delivery in cancer therapy. Exosomal delivery of adriamycin and paclitaxel that has been used in targeted cancer therapy with less toxic side effects and immunogenicity , 75. In Tumor stem cells are believed to be the seed cells of the primary cancer and the source of drug resistance to radiotherapy and chemotherapy. The accurate delivery of drugs to tumor stem cells is the current focus and challenge facing cancer therapy. Nanotechnology combined with exosomal drug delivery has the potential to address this challenge and potentially improve the efficacy and specificity of targeted cancer stem cell therapy . The exoin vivo studies for the treatment of breast cancer and colorectal cancer have shown that these exosomes enhance cell uptake through clathrin-dependent and independent endocytic pathways, significantly improve the cytotoxicity of aspirin to breast cancer and colorectal cancer cells, and enhance tumor cell apoptosis and autophagy. In addition, aspirin encapsulated by nanocrystalline exosomes has an unprecedented ability to remove tumor stem cells (Exosomes may also provide solutions to long-standing drug delivery challenges. While aspirin was found to have anti-tumor effects, the challenge of delivering it to the tumor has limited its application . In ordeem cells  (FigureExosomes are extracellular vesicles that contain nucleic acids, proteins, lipids and other substances. Compared with tumor markers that exist in tissues and body fluids, exosomes are not only highly stable but are also rich in content, which lays a solid foundation for the future clinical application of exosomes . The extExosomes are versatile and serve a critical function in intercellular communication. In this article, we elaborated on exosome-mediated tumor metastasis, tumor angiogenesis, EMT, exosome immunomodulatory functions, and the role of combining nanoengineering technologies to fight cancer. Future studies may focus on the potential heterogeneity of tumor cell-derived exosomes, which will help to understand the clonal expansion of tumor cells and the characteristics of clonal expansion after cancer treatment . By usinAs an intermediate of intercellular communication, exosomes play a vital role in the pathogenesis, diagnosis, and treatment of tumors. However, there are still a number of problems that need to be addressed before its clinical application. First, how do we identify exosomes? To address this problem, a relevant study has reported that Alix, CD9, TSG101, and CD63 are protein markers of exosomes . HoweverThe ultimate service of basic medicine is the patient. According to the clinical needs, how to extract exosomes quickly and effectively and apply them to the diagnosis and treatment of cancer patients should be a priority to the field and a major objective of future research. In addition, a number of unknown molecular mechanisms, such as how exosomes mediate cancer progression and tumor suppression, and immature artificial exosome synthesis techniques have brought new challenges to exosomes in the diagnosis, treatment, and prognostic guidance of cancer.YL wrote the essay. KS and YC made tables. YL, XW and ZC drew diagram. KC and YT revised the introduction and the first half of the article. JL helped to revise the conclusion. XC and JZ helped to revise the framework of the article. All authors contributed to the article and approved the submitted version.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.


Walking function recovery in spinal cord injury (SCI) is tackled through several therapeutic approaches in which precise evaluation is essential. A systematic review was performed to provide an updated qualitative review of walking ability outcome measures in SCI and to analyze their psychometric properties. PubMed, Cochrane, and PEDro databases were consulted until 1 April 2020. Seventeen articles written in English were included. Five of them studied the walking index for SCI, four studied the 10 meter walk test, and two studied the six-minute walk test, the timed Up and go test, and the Berg balance scale. The rest of the articles studied the following metrics: gait profile score, spinal cord injury functional ambulation profile, five times sit-to-stand test, spinal cord injury functional ambulation inventory, spinal cord independence measure (indoors and outdoors mobility items), locomotor stages in spinal cord injury, community balance and mobility scale, and activity-based balance level evaluation scale. The choice of a single or a set of metrics should be determined by the clinician. Based on the results obtained in this review, a combination of outcome measures is proposed to assess walking ability. Future work is required to integrate a more realistic environment for walking assessment. Annually, an estimated 250,000–500,000 individuals suffer a spinal cord injury (SCI) worldwide . The conWalking function recovery is tackled through several pharmacological, physical, robotic, and neurophysiological approaches in which precise evaluation of walking function is mandatory . It is tIOMob), and the locomotor-related items of the functional independence measure (FIML). Similarly, with the aim of identifying the most clinically relevant outcome measures of general motor function after traumatic SCI, Labruyère et al.  AND  AND ; following an advanced search in the field “Title, Abstract” or “Title, Abstract, Keyword”.The included articles fulfilled the following inclusion criteria: (i) studies including walking ability outcome measures in SCIs, also including balance; (ii) obtained by instrumental, numerical, or observational procedures; (iii) the latest version if there was more than one; (iv) analysis of at least one psychometric property of the measure; (v) no limitation on American Spinal Injury Association (ASIA) Impairment Scale (AIS) or Neurological Level of Injury (NLI) ; (vi) adThis systematic review excluded articles according to the following exclusion criteria: (i) studies published as conference proceedings and clinical trials registration; (ii) studies that used walking ability outcome measures to evaluate the results of an intervention but did not analyze their psychometric properties; (iii) systematic or no systematic reviews.General characteristics of the studies, including sample size, injury features , population characteristics (age and gender), inclusion and exclusion criteria followed in the study, walking ability outcome measures studied, and statistical results of psychometric properties analyzed , were extracted. The evaluation criteria for defining the measurement properties and their standard values are shown in The screening of titles and abstracts obtained from the electronic search was followed by one reviewer (IS-A), who decided which articles met the inclusion criteria. For those included, the full-text articles were obtained, and two reviewers (IS-A and AJd-A) executed a new screening to confirm their relevance and to remove those that were not of interest to the review. Any disagreement on the selection of the articles was resolved by discussion with a third author (RC-d-l-C).The initial search of the databases yielded 1016 results, and five additional records were identified through other sources (website searches and citation tracking). After duplicate removal, 846 articles were screened applying the inclusion and exclusion criteria by reading the titles and abstracts. Eight hundred and eight articles were excluded and 38 were full-text screened for eligibility. Twenty-one articles were excluded  because IOMob, the locomotor stages in spinal cord injury (LOSSCI), the Berg balance scale (BBS), the community balance and mobility (CB&M) scale, and the activity-based balance level evaluation (ABLE) scale. The search yielded three more outcome measures that were excluded due the lack of psychometric data, the Gillette gait index (GGI) and the 50FWT, or because its mobility sub-scale broadly measured ambulation and wheelchair propulsion, i.e., the FIML-.A total of 13 walking ability outcome measures were obtained and grouped into different categories : the gaiFive out the 17 articles analyzed the WISCI, four the 10MWT, and two the 6MWT, TUGT, and the BBS. Each one of the remaining walking ability outcome measures were analyzed in only one article . The WISThree-dimensional gait analysis (3DGA) is considered the gold standard to evaluate walking abnormality and to assess changes after rehabilitation interventions .Accordingly, the GPS is a single index outcome measure derived from 3DGA data that summarizes the overall quality of a patient’s kinematics by quantifying its deviation from a reference population without gait pathology . The GPSThe 10MWT measures the time invested in walking 10 m at a preferred or maximum walking speed  with phyThe SCI-FAP  encompasThe TUGT  measuresThe FTSST records the time needed to, from sitting, stand up and sit back five times at the safe fastest speed without using external supports . It has The 6MWT measures the distance recorded during six minutes walking at a preferred or maximum walking speed . It was The WISCI is a walking scale specifically developed for the iSCI population. It was originally composed of 19 levels , but a lThe SCI-FAI is an observational gait assessment instrument aimed at measuring functional walking ability in individuals with SCI through three categories of performance : (i) gaiIOMob, which assess walking ability, is classified in three main assessment groups: mobility indoors, mobility for moderate distances (0–100 m), and mobility outdoors (>100 m). All of them are similarly scored, from the requirement of total assistance (scored 0) to the independence of AADs, orthoses and personal supervision (scored 8).The SCIM is a global disability scale developed by Catz et al.  for indiThe LOSSCI is a five-stage scale result of applying and adapting to SCI  the origThe BBS is a 14 item scale, originally designed to assess balance and fall risk in elderly population , that coThe CB&M scale, originally developed and validated for the brain-injured population , is a 13The ABLE scale, developed and tested to assess balance activities in population with SCI , was iniThere are several outcome measures used in clinical settings to assess walking ability and/or evaluate the effects of walking rehabilitation interventions in people with SCI. In the last decade, several authors conducted systematic reviews in the field of outcome measures used to assess disability , generalTo be easily and broadly applied in clinics, the assessment procedures must be timely, affordable, and with no need of sophisticated, thus complicated, equipment. The evaluator has to choose the metric, or set of metrics, within all the broad range of possibilities for measuring changes in walking ability, including balance, which may be challenging due to the number, heterogeneity, and differences in construct of the available metrics. Psychometric properties of each outcome measure, assessment time, cost of the specialized equipment, and human resources required, not only to accomplish the test but also to analyze the results, are relevant aspects that may determine the choice. Furthermore, some metrics seem to be redundant. In this sense, this paper aimed to bring a qualitative review of the different walking outcome measures which are currently available to measure balance and ambulation in people with an SCI to provide a useful and reliable guide in clinical and research settings.With regard to the outcome measures included in this review, 3DGA provides specific information to guide rehabilitation interventions to improve walking function of people with traumatic and non-traumatic iSCIs, quantifying changes in gait kinematics and, hence, the impact on the kinematics of the intervention . ConcernThe 10MWT and 6MWT allow to assess functional ambulation in which gait speed over short distances (10MWT) is thought to represent crossing the street, while longer distances (6MWT) reflect endurance required for community ambulation . Both teConcerning the TUGT, since it encompasses a more complex timed set of tasks, such as standing up, walking, turning and sitting down, it might better reflect community walking ability . The TUGWhile the previous spatiotemporal-related walking and balance measures can be applied in a simplified yet standardized environment, the SCI-FAP evaluates walking performance on a variety of common walking skills which are representative of real-life scenarios . FurtherThe WISCI is a widely used scale in SCIs in which levels are ordered by degree of the underlying person’s impairment, from most impaired to least impaired , consideIOMob has shown to be a valid and responsive outcome measure to assess the efficacy of new interventions on ambulatory function in people with SCI [IOMob ranks in terms of levels of independent walking in which independency from AADs is ranked higher in comparison with the WISCI [Regarding the SCIM, since it is a disability scale for people with SCI, it is not a specific scale for ambulation. Nevertheless, the SCIM IIwith SCI . Furtherhe WISCI . Future The SCI-FAI is a valid, reliable, and sensitive outcome measure of walking ability for individuals with SCI  that attAs alternative tool to all the walking ability outcome measures above mentioned, the LOSSCI allows to assess the locomotor progress of a person with SCI from an ontogenic point of view, and it has shown to be a valid and reliable outcome measure . ConcernFinally, balance assessment has been classically ignored as an essential component of ambulation in people with SCI. Besides the TUGT and the FTSST, which involve a balance component, there is not a wide range of balance outcome measures in the literature evidencing their validity in SCI. The most popular is the BBS, which has shown to be a valid ,29 and rWalking ability is a complex set of functional tasks involving static and dynamic balance, joint kinematics, limb coordination, changes of speed, and endurance requirements to allow the person adapting to a changeable environment present in real-life situations. From this standpoint, it does not make sense to divide walking assessment in different outcome measures but evaluating walking function with a single outcome measure able to capture walking complexity as a whole. Nevertheless, the findings of this review showed several outcome measures which assess different relevant aspects involved in walking ability from an isolated point of view. Therefore, the authors suggest combining several metrics, without redundancy, to provide a global assessment of walking function.Since 3DGA is the gold standard to measure improvements in walking function quantifying changes in gait kinematics , kinematIn any case, a selection and a combination of outcome measures to assess walking and balance in people with SCI should be a choice of the clinician according to the purpose of the assessment, the level of walking function of the person evaluated in pursuit of avoiding ceiling or floor effects of the metric, time invested in performing the test, and technical and human resources required.The main strength of this work is the contribution with an updated review of all the outcome measures available in the published literature to assess walking ability in SCI, considering the diverse factors involving walking function and their limitations, and analyzing their psychometric properties. Nevertheless, this systematic review presents some methodological limitations: (i) articles included were not classified according to the levels of evidence and grades of recommendation for diagnosis studies established by the Oxford Center for Evidence-Based Medicine ; (ii) meThis paper provides an updated review of the walking ability outcome measures that are currently available to measure ambulation in people with SCI, their psychometric properties, and their limitations. The choice of a single or a set of outcome measures will be determined by the clinician depending on the purpose of the assessment and the financial, human, and time resources required. New outcome measures of walking ability , not envisaged in previous systematic reviews in the published literature, should be considered to assess walking ability in individuals with SCI, considering thus different functional perspectives related to more realistic daily life situations. Since walking ability is a complex set of functional tasks, the authors propose a combination of 3DGA with a minimum of valid and reliable outcome measures, such as the 10MWT, TUGT, and WISCI, to cover the whole functional spectrum of walking ability. Future work is required to validate multivariate walking metrics such as the GPS, and to integrate SCI-FAP tasks in the 3DGA protocol, providing a more realistic environment for walking assessment.


Metabolic reprogramming has been suggested as a hallmark of cancer progression. Metabolomic analysis of various metabolic profiles represents a powerful and technically feasible method to monitor dynamic changes in tumor metabolism and response to treatment over the course of the disease. To date, numerous original studies have highlighted the application of metabolomics to various aspects of tumor metabolic reprogramming research. In this review, we summarize how metabolomics techniques can help understand the effects that changes in the metabolic profile of the tumor microenvironment on the three major metabolic pathways of tumors. Various non-invasive biofluids are available that produce accurate and useful clinical information on tumor metabolism to identify early biomarkers of tumor development. Similarly, metabolomics can predict individual metabolic differences in response to tumor drugs, assess drug efficacy, and monitor drug resistance. On this basis, we also discuss the application of stable isotope tracer technology as a method for the study of tumor metabolism, which enables the tracking of metabolite activity in the body and deep metabolic pathways. We summarize the multifaceted application of metabolomics in cancer metabolic reprogramming to reveal its important role in cancer development and treatment. In recent years, metabolism, as an important link between environmental factors, metabolic small molecules, host genes and diseases, has gained increasing interest regarding its relationship with tumors. Under environmental selection pressures induced by microenvironmental and genetic factors, the inside of the tumor undergoes evolution. Simultaneously, under the control of genotypes, metabolic characteristics of the tumor undergo adaptive changes; this is termed metabolic reprogramming . StudiesMetabolic reprogramming of the tumor microenvironment is considered one of the markers of cancer and an important direction of tumor research . ContinuMetabolomics techniques are defined as the measurement of the dynamic multiparameter metabolic response of biological systems to various stimuli and genetic changes in specified quantities . They prMass spectrometry-based technology has become the mainstream technology for the analysis of targeted metabolomics pathways. Numerous studies have revealed that differences in small molecular metabolites, such as serum, tissues, urine, and saliva, and changes in corresponding metabolic pathways are closely related to tumor risk, tumor type, and the sensitivity and efficacy of chemotherapy drugs as well as potential drug targets . MetabolDuring cancer development, metabolic reprogramming provides cancer cells the ability to survive and proliferate. The most famous is the Warburg effect, which suggests that the aerobic glycolysis pathway is closely related to the occurrence of cancer. In addition, deregulated anabolism/catabolism of fatty (FAs) and amino acids, especially glutamine, serine, and glycine, have been shown to function as metabolic regulators in supporting cancer cell growth. The occurrence and development of cancer cells are closely related to the three metabolic pathways. Owing to the need for malignant proliferation, tumor cells exhibit a rapid glycolysis phenomenon in various environments called the Warburg effect. Tumor cells have a high ability to proliferate and a high demand for energy, which often leads to hypoxia in the local tissue microenvironment . AlthougHigh-throughput omics screening has shown that the tumor microenvironment and various cancer-promoting signaling pathways significantly up-regulate the glycolysis process, which results in the Warburg effect. Recent investigations of renal carcinoma cells and tissues from xenografted mice and patients using metabolomics, proteomics, and transcriptomics have unambiguously confirmed that this phenomenon is a key component of metabolic reprogramming . As an eEssentially, the Warburg effect in tumor cells is caused by the increased expression of metabolic enzymes related to the glycolysis pathway. In recent years, the regulation of glycolytic metabolic enzymes, especially rate-limiting enzymes, has attracted considerable attention in the field of oncology . ThroughTumor cells exhibit metabolic plasticity, which provides a selective advantage for the survival and proliferation of tumor cells in harsh microenvironments, such as hypoxia, acidosis, and malnutrition . SignifiLipid metabolism presents as a network of pathways with flexible feedback loops and crosstalk to meet the increased metabolic needs of cancer cells. Based on multi-omics data of pan-cancer, researchers have found extensive alterations in FAs, arachidonic acid, cholesterol metabolism, and peroxisome proliferator-activated receptor (PPAR)-signaling in different tumors, and lipid metabolism features are shared among tumors with similar tissue origin tumors. Moreover, possible causes of metabolic disorders have been correlated with lipids in tumors from several perspectives, which include somatic mutation, DNA methylation abnormality, and regulation of transcription factors . For exaLipid metabolism can not only affect the growth of tumor cells by metabolic recombination of fatty acids and other molecules, but also regulate the cross-talk between tumor cells and tumor associated stromal cells to modulate the high metabolic needs of the tumor. It has been reported that the liver X receptor (LXR), a lipid activated transcription factor, plays an important role in modulating the TME. Apoptotic tumor cells containing oxysterols activate LXR in macrophages causing suppression of dendritic cell migration and recruitment of neutrophils in tumors resulting in tolerance and immunosuppression . Cancer Cancer cells’ need for amino acids increases in order to sustain their rapid proliferation. In addition to their use in protein synthesis, amino acids are increasingly being studied as metabolites and regulators that support cancer cell growth . MultiplIncreased glutamine metabolism is a commonly observed metabolic change in cancer, and glutamine is second only to glucose in importance as a nutrient in cancer. As the most abundant free amino acid, glutamine is involved in a series of energy generation, macromolecular synthesis, and signal transmission pathways of cancer cells by providing nitrogen and carbon atoms . GlutamiOther amino acids, such as aspartic acid and arginine, are involved in the reprogramming of amino acid metabolism in cancer. In a study that investigated significant metabolomic changes in plasma in the early and late stages of 4T1 metastatic breast cancer in mice, the plasma arginine concentration was higher during the early stage of metastasis but gradually decreased, and the urine-arginine/arginine ratio increased in the late stages . This isWith the comprehensive development of modern molecular biology, many new tumor markers carry important clinical value for the early diagnosis and screening of malignant tumors; however, current routine tumor markers lack sensitivity and specificity for the early diagnosis of tumors. As an emerging omics technology, metabolomics mainly involves the study of small molecular metabolites  and reflects a series of small changes in the body at the level of metabolites, which is suitable for the diagnosis of diseases. Many scholars have used a variety of detection techniques to conduct research on the early diagnosis and treatment prediction of tumors . MetabolBecause blood is a readily available biological specimen, blood biomarker studies account for the majority of metabolomics and tumor studies. The application of metabolomics technology has led to significant breakthroughs in the discovery of biomarkers for a variety of tumors, which include pancreatic, liver, lung, and breast cancers . For insIn the case of pancreatic cancer, researchers have identified five new metabolic biomarkers  that can be used for the clinical diagnosis of pancreatic cancer by comparing plasma metabolomics between patients with pancreatic cancer (n = 60) and healthy controls (n = 60) . SubsequThe non-invasive nature of urine biomarkers makes them particularly suitable for a wide range of screening purposes, especially for measuring urine from asymptomatic high-risk groups to distinguish between those who may and may not be carrying a disease. Accurate and effective analyses of urine metabolites offer promise for their applications to provide a deeper understanding of tumor pathology and eventually, clinical transformations .In urological tumors, Ganti used GC-MS to measure carnitine in two different laboratories  and showed that differences in urinary concentrations of several acylcarnitines were a function of cancer status and renal cancer grade. Most cancer patients and those with high-grade cancers have increased acylcarnitine in their urine . In a blIn addition to blood and urine, other biological fluid biomarkers are available for the early diagnosis of specific cancers. Saliva is a biological fluid made up of more than 99% water and less than 1% of proteins, electrolytes, and other low-molecular-weight components . Saliva Since then, several studies have shown that the combination of saliva and tumor metabolomics is beneficial for the identification of salivary metabolite biomarkers and the screening of noninvasive oral cancers . In addiCerebrospinal fluid (CSF) is a biological fluid that is most likely to be affected by central nervous system dysfunction, and its analysis can better reflect inherent neurological and biochemical changes . In 2012In recent years, several studies have also confirmed that the use of untargeted metabolomics techniques to analyze metabolic characteristics of different brain tumors in clinical CSF samples enables reliable identification of significant metabolic differences between different brain tumors, which offers significant promise for diagnoses of brain tumors . More meThe main purpose of drug therapy of tumors is to control the growth of the tumor and improve the quality of life of patients. Selecting the most effective anti-tumor drugs has become a top priority. In clinical practice, metabolomics can be used to detect body or cell metabolites that reflect the effects of anti-tumor drugs on the body or cells to improve the efficacy of drugs and reduce avoidable adverse reactions.Kim et al. used NMR to investigate metabolic alterations following adriamycin (ADR) treatment for gastric adenocarcinoma. After human gastric adenocarcinomas were implanted into mice, ADR was intraperitoneally administered for 5 days, and urine was collected on days 2 and 5. Results showed that the levels of trimethylamine oxide, hippurate, and taurine decreased in the tumor model and increased following ADR treatment. In addition, the levels of 2-oxoglutarate, 3-indoxylsulfate, trigonelline, and citrate, which all increased in the tumor model, significantly decreased to those of normal controls following ADR treatment . In a plFurthermore, using untargeted lipidomics and quantitative polymerase chain reaction, Zhang et al. identified distinct features of lipid metabolism in imidazole ketone erastin (IKE)-induced ferroptosis and demonstrated that IKE slows tumor growth . Once drThe metabolic pattern of tumor cells changes following the development of drug resistance. The same drug can produce different metabolic changes in sensitive and drug-resistant cells . TherefoMetabolomics can be used to distinguish between platinum resistance and metabolite levels. Poschner et al. used LC-MS to characterize the levels of steroids, active estrogen, and sulfated or aldehyde glucose during the development of platinum resistance in ovarian cancer and found that these metabolites are highly expressed in carboplatin-sensitive cells . In a st13C6]-Glc, into biological systems and detecting subsequent metabolic transformations. Incorporating stable isotopes, such as 2H, 13C, or 15N, into biological precursors has long been used to trace their metabolism in living systems. . Biological samples can be studied using NMR, mass spectrometry, and other detection platforms -glucose tracer -pyruvate in order to apply as a tool for imaging liver cancer (In recent years, Dynamic nuclear polarization enhanced magnetic resonance imaging based on isotope tracer have become dependable imaging tools for the diagnosis and treatment assessment in cancer. Based on preclinical studies that have demonstrated the use of hyperpolarized (1–13C] -pyruvate imaging tools for prostate cancer, Researchers have investigated the r cancer . Applicar cancer .By gaining insights into metabolic dysfunction due to cancer development or drug interventions, Metabolic flux analysis and fluxomics can be integrated with genomic and proteomic information to achieve systems biochemical insights in both model systems and individual human patients.Metabolomics has been used across many aspects of cancer research, which include cancer pathophysiology, biomarker discovery, and therapeutic response. Metabolic reprogramming is a hallmark of malignancy, and changes in metabolic profiles strongly influence cancer development, progression, and response to treatment. Metabolomics techniques can be used to monitor the dynamics of tumor metabolism and response to treatment over the course of the disease. Moreover, another area of increasing importance is the identification of biomarkers for personalized treatment strategies. At the same time, metabolomic analysis may also yield more accurate and useful clinical information about the metabolic needs of the tumor, as well as the identification of new pharmacodynamic biomarkers and the monitoring of drug resistance of the tumor. On this basis, the applications of stable isotope tracer technology to the field of metabolomics have become an important part of biological research to track the activity of metabolites in the body and trace deep metabolic pathways.However, there are still several limitations in the study of metabolic reprogramming in tumors using metabolomics. Methodologically, absolute homogeneity across different batches of biological samples cannot be achieved, and the techniques and methods used should be optimized to establish a set of highly sensitive routine procedures that apply to metabolites of varying polarity across different samples. Secondly, most existing studies focus on the metabolomics of biological fluids or extracellular metabolites; however, it is also of importance to study the metabolic characteristics of intracellular metabolites and cancer cells. Thirdly, the mechanisms underlying metabolic changes need to be clarified. This can be achieved by integrating transcriptomic and/or proteomic analyses to identify genes or proteins that cause or are associated with metabolomic changes, which may be potential targets for tumor therapy. Analyzing metabolic changes in tumor cells in response to drugs and revealing the metabolic mechanism underlying tumor drug resistance may provide opportunities to overcome tumor chemotherapy resistance or reverse tumor sensitivity via metabolic regulation. Metabolomics provides a novel approach to study the metabolic reprogramming of tumors and will continue to be widely used in the diagnosis and treatment of different tumors in the future.


Besides the applications as a new in vitro model to study the physiological characteristics of normal tissues and organs, organoids are also used for in vivo cancer cell characterization, anti-cancer drug screening, and precision medicine. However, organoid cultures are not without limitations, i.e., the lack of nerves, blood vessels, and immune cells. As a result, organoids could not fully replicate the characteristics of organs but partially simulate the disease process. This review attempts to provide insights into the organoid models for cancer precision medicine.As organ-specific three-dimensional cell clusters derived from cancer tissue or cancer-specific stem cells, cancer-derived organoids are organized in the same manner of the cell sorting and spatial lineage restriction   However, due to the significant differences between rodents and human, it is impossible to translate most of the findings from mouse models to human. The patient cancer-derived organoids (PCDOs) could be a possible answer to that need Although a global health threat, research on the pathogenesis and treatment of cancer is still ongoing, in which two-dimensional (2D) cell culture and xenotransplantation of cancer cells provide the materials for experiments in vitro tissue or organ analogs derived from patient cancer tissue or cancer-specific stem cells cultured using the self-renewal and differentiation ability of stem cells PCDOs are small in vitro without a mesenchymal niche In 1907, Wilson demonstrated for the first time that mechanically isolated sponge cells could regroup and self-organize to form a complete organism in vivo and is conducive to cell culture and differentiation in vitro[38]. Epidermal growth factor, basic fibroblast growth factor, insulin and transferrin, and dexamethasone and Wnt activator regulate the formation of organoids and cell differentiation The commonly used organoid culture system is matrix glue. The basement membrane matrix is extracted from mouse sarcoma rich in extracellular matrix protein in vivo and help to study the morphological characteristics and organ transplantation Organoids could derive from one or more stem cells, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and adult stem cells (ASCs) In 2009, the first PCDOs model was established with small intestinal crypt stem cells in vivo. PCDOs could simulate the pathological characteristics, development process, and biological signals of cancers PCDOs could retain the biological characteristics and heterogeneity of cancer tissue and have a stable genome and short culture cycles after multiple passages, making it an ideal model for cancer research in vitroThe tumor microenvironment (TME) comprises cancer cells, stromal cells, immune cells, and endothelial cells Tumorigenesis is the result of gene mutation accumulation ,79. The Besides the application of examining normal development, organoids have also been used to study tumorigenesis. In most studies on cancer that adopt organoids, primary carcinoma samples have been generated under ASC-organoid conditions. However, CRISPR mutagenesis technology has been applied to PSC-based organoids to generate cancer-causing mutations, for example, to model human brain tumors. In addition, Fine and colleagues have explored PSC-derived mini-brains as an environment for growing patient-derived glioblastoma cells Jager et al. Cancer is a highly heterogeneous disease The cost of isolating and culturing the cancer patient organ samples and constructing the PCDO biological sample bank is relatively low; the time spent is short; thus, PCDOs are more suitable for gene operation than the cancer xenotransplantation model Organoids could derive from both cancer tissues and normal adult tissues Currently, therapeutic drugs for different stages of cancer are being developed rapidly. However, prescribed therapy is usually based on the general success rate of the drug rather than the response of specific patients to the drug. Moreover, drug testing with conventional monoculture pre-clinical models is misleading and most likely responsible for the high failure rate of phase 3 trials. It is, therefore, a clinical and research priority to construct a reliable model that can predict patient responsiveness and enable patient-tailored treatment strategies. Although the development of disease staging, pathological typing, gene sequencing, and molecular typing is helpful to guide the clinical treatment of cancer patients, effective tools are still needed to support the prediction of drug response of specific individuals Compared with traditional precision medicine, such as gene expression detection and drug sensitivity test, the application time of cancer organoids is significantly shorter, facilitating the real-time evaluation of the effect on each patient Seidlitz et al. in vivo. The interaction between host and pathogen could be simulated by the microinjection of pathogenic microorganisms into the epithelial cavity of the organoids. Co-culture of PCDOs with host immune cells and other cells can provide convenient conditions for revealing the role of the TME in tumorigenesis and development. Compared with traditional cancer cell modeling methods, PCDOs could maintain genomic stability and cancer heterogeneity, which provides a model closer to the characteristics of the original cancer tissue.In cancer research, PCDOs could effectively simulate the dynamic pathological changes of cancer cells The emergence of patient-derived xenograft (PDX) models as a surrogate, translational, and functional representation of the patient with cancer has led to the advances in drug discovery and testing of novel targeted approaches and combination therapies. However, current established PDX models fail to represent the diverse patient population and, most importantly, the specific ethnic patient populations that have higher rates of incidence and mortality. When compared with the cancer xenotransplantation model, cancer organoids have a lower cost and are less time-consuming. In addition, establishing a PCDO biological sample bank could provide an optimization model for clinical drug high-throughput screening and precision medicine and contribute to research cooperation among different countries, research institutions, and teams.in vitro models, there are still limitations and shortcomings. The establishment of PCDOs requires a variety of technical and logistical support. The technical requirements for establishing a PCDO sample library by separating the cancer tissue of patients are high; obtaining enough cancer samples is difficult due to the limited number of patients and the fact that samples cannot be obtained by surgery from patients with advanced cancer. In TME, there are cancer tissue and many supporting cells, stroma, and neovascularization, which are involved in cancer growth and metastasis. PCDOs were cultivated in the best growth environment and growth-promoting matrix and lack angiogenesis, so they can not completely simulate the TME of cancer tissue in vivo. Further technical optimization is needed based on PCDOs culture in future studies to provide the best model for revealing the occurrence and development mechanism of cancer, thereby leading to the development in cancer prevention, treatment drugs, and precision medicine.Although PCDOs are unique and advantageous in vivo. Up to now, researchers have successfully produced a variety of PCDOs, including colon cancer, prostate cancer, gastric cancer, breast cancer, pancreatic cancer, ovarian cancer, kidney cancer, and bladder cancer. As a new type of in vitro model, organoids maintain the characteristics of cancer cells in vivo and could be used to study the physiological characteristics of normal tissues and organs. PCDOs could also be used for anti-cancer drug screening and precision medicine.PCDOs recapitulate the spatial arrangement of the original tissue and simulate the characteristics of cancer and the heterogeneity of cancer cells The authors declare that they have no competing interests.