Tumor progression involves a series of biologically important steps in which the crosstalk between cancer cells and the surrounding environment is an important issue. Angiogenesis is a key tumorigenic phenomenon for cancer progression. Tumor-related extracellular vesicles (EVs) modulate the tumor microenvironment (TME) through cell-to-cell communication. Tumor cells in a hypoxic TME release more EVs than cells in a normoxic environment due to uncontrollable tumor proliferation. Tumor-derived EVs in the TME influence endothelial cells (ECs), which then play multiple roles, contributing to tumor angiogenesis, loss of the endothelial vascular barrier by binding to ECs, and subsequent endothelial-to-mesenchymal transition. In contrast, they also indirectly induce tumor angiogenesis through the phenotype switching of various cells into cancer-associated fibroblasts, the activation of tumor-associated ECs and platelets, and remodeling of the extracellular matrix. Here, we review current knowledge regarding the involvement of EVs in tumor vascular-related cancer progression. Extracellular vesicles (EVs) were first discovered in 1967 and initially called “platelet dust,” which was defined as subcellular coagulant materials . ThereafIt is clear that the majority of cancer-related mortalities are the result of cancer metastasis . Cancer The TME is more hypoxic than the normal internal environment due to the uncontrollable growth of cancer cells . Solid tSolid tumor cells near functional blood vessels, in which the normoxic area has an oxygen level of 38 mmHg (5%) , are viaTumor cells in a hypoxic environment produce more EVs than cells in a normoxic environment, and this environment substantially alters the protein and nucleic acid profiles of EVs . EVs indSeveral studies have shown that tumor cells release more EVs under hypoxic conditions than under normoxic conditions in a hypoxia inducible factor (HIF)-dependent manner. HIFs have various effects on cellular functions and control various types of oxygen responsive target genes. HIF-1α induces various angiogenetic growth factors, including vascular endothelial growth factor (VEGF), stromal derived factor 1 (SDF1), angiopoietin 2, placental growth factor, platelet derived growth factor (PDGF), and stem cell factor . These fThe release of MVs or EVs is promoted by the HIF pathway. Dimethyloxalylglycine (DMOG), which inhibits HIF hydroxylase, mediates the activation of hypoxic signaling and results in a significant increase in EV release . AnotherHypoxia induced by tumor growth further limits cell proliferation. However, tumor cells in hypoxic areas produce EVs, which have several effects on the stroma. Additionally, EVs derived from hypoxic tumor cells influence important steps in cancer metastasis, such as angiogenesis. The HIF pathway is one of the most important pathways for tumor cells to survive in a hypoxic environment and mediates tumor angiogenesis in the TME. EVs containing HIF-1α can also induce tumor angiogenesis and metastasis.Endothelial cells play essential roles in microvessel sprouting and angiogenesis associated with the TME and cancer metastasis. Angiogenesis is defined as the formation of new blood vessels from the existing vasculature to supply nutrients and oxygen to hypoxic organs . VEGF isTumor cells promote the loosening of intercellular adhesions between ECs by impairing adhesion molecules and gap junctions. Promotion of vascular permeability is related to enhanced cancer growth and metastasis by supplying enough nutrients and oxygen to growing tumors and promoting intravasation and extravasation. Accumulating evidence has revealed that tumor-associated EVs can modify vascular permeability . miR-23ain vivo  and disrupts the blood-brain barrier and consists of tight junctions between ECs and surrounding cells that contribute to low permeability and promote cancer metastasis in distant organs . Anotherin vivo . ECs essTumor-derived EVs can also enhance tumor lymphangiogenesis and lymph node (LN) metastasis. It has been reported that different types of human cancer promote lymphangiogenesis, which is correlated with LN metastasis, and the presence of LN metastasis often reflects a poor prognosis . VEGF-C in vitro and enhanced tumor lymphangiogenesis and LN metastasis in vivo through the upregulation of prospero homeobox 1 (PROX1). Another study showed that cervical squamous cell carcinoma-derived EVs promoted LEC migration and tube formation in vitro and facilitated lymphangiogenesis and LN metastasis in vivo  also secrete EVs, and several studies have suggested that these EVs contribute to angiogenesis and metastasis. ECs in a hypoxic environment secrete EVs, which include lysyl oxidase-like 2 (LOXL2) . LOXL2 iin vivo . In addiin vivo .Data from several clinical studies have suggested that EV-derived ECs are involved in cancer prognosis. In patients with lung cancer, circulating levels of endothelial-derived EVs, especially MVs, are independent predictors of mortality . In breaEpithelial cells in tumors can change their phenotype, downregulate epithelial characteristics and acquire mesenchymal characteristics . This ceEndothelial cells in the TME can also modify their endothelial phenotype switch to the mesenchymal profile in a manner similar to that of epithelial cells. This phenotype switch in ECs is referred to as endothelial-to-mesenchymal transition (EndoMT) . RecentlThe phenotypic switch in ECs via tumor-related EVs is thought to contribute to the intravasation and extravasation of tumor cells via an impairment in vascular permeability and becomes a resource for CAFs . EndoMT Cancer-associated fibroblasts are the main component of stromal cells in the TME. They originate from various types of cells, such as epithelial cells, resident fibroblasts, bone marrow derived from mesenchymal stem cells, hematopoietic stem cells, and ECs . MyofibrEvidence of the mechanism by which stromal cells are activated by tumor cells via EVs is gradually accumulating . AccordiCancer cell-derived EVs contribute to the conversion of fibroblasts into CAFs in the premetastatic niche. In HCC, miR-1247-3p is carried to the premetastatic niche in the lung through EVs and converts fibroblasts to CAFs by decreasing the expression of its target, β-1,4-galactosyltransferase III (B4GALT3), to activate the β1-integrin-NF-κB pathway . IntegriNot only cancer cells but also CAFs secrete EVs, which may stimulate cancer metastasis and angiogenesis. The ECM consists of collagen, proteoglycans, elastin, fibronectin and integrins that provide structural and biochemical support for cancer cell growth . The ECMRecently, relevant studies in which tumor-derived EVs containing MMPs were associated with angiogenesis have gradually accumulated . The invin vitro and in vivo  is a key therapy for many tumor types and a well-established research area . AATs heOne of the mechanisms by which AATs function is vascular mimicry (VM) . In 1999in vivo (Vascular mimicry can be induced by a hypoxic environment. Several studies have suggested that HIF-1α is involved in VM . The expin vivo . Furtherin vitro and enhanced endothelial vasculogenesis and VM in vivo (On the other hand,  in vivo . This meThese studies indicate that EVs in the TME are implicated in VM after AATs, resulting in AAT resistance. If VM can be prevented, AATs will be beneficial to patients with cancer. The prevention of EV release in TECs induced by AATs can be one of the target therapies for VM and AAT resistance. Another study showed that miR-124 inhibit VM formation of cervical cancer through inhibition of MMP-2, MMP-9, and VEGF . miR-9 aA hypoxic environment due to the rapid and uncontrollable growth of tumor cells influences tumor cells and stromal cells to secrete not only several chemical mediators but also EVs, which contribute to neovasculature and metastasis. Neovasculature is one of the most important steps in metastasis and components of the TME. Tumor and stromal cells communicate with each other via EVs in the TME. Tumor-derived EVs induce angiogenesis in ECs in a VEGF-dependent or VEGF-independent manner. EVs secreted by tumor cells impair EC adhesion molecules and gap junctions to promote vascular permeability. The loss of endothelial markers and the acquisition of mesenchymal markers is referred to as EndoMT, which is activated by tumor-related EVs, contributes to the intravasation and extravasation of tumor cells and becomes a resource for CAFs. CAFs are derived from various types of cells and secrete EVs related to angiogenesis. Several types of cells, such as ECs, CAFs, platelets, and tumor cells, secrete EVs containing MMPs and result in ECM remodeling and angiogenesis. Platelets educated by tumor cells secrete PMPs that induce inflammation and coagulation. PMPs are the most abundant EVs in the blood and are associated with tumor angiogenesis, VTE, and patient prognosis. AAT is an important therapy for tumor progression. However, tumor cells induced by AATs establish capillary forms without vascular-related cells; this is referred to as VM.As mentioned in this review article, we provide evidence that tumor and stromal cells are involved in tumor neovasculature through crosstalk with each other via EVs. A number of studies have suggested that tumor cells rearrange surrounding cells and the ECM using EVs, and these studies have helped understand the mechanism of tumor angiogenesis. In addition, recent studies on the involvement of non-malignant cells in cancer progression and regression have gradually accumulated.The function of EVs derived from tumor cells and stromal cells may shed light on the mechanisms of cancer angiogenesis and progression. Tumor-derived EVs may also contribute to tumor cells becoming resistant to anti-tumor therapy, such as VM induced by AATs. Therefore, if we discover a method to prevent the release of EVs associated with tumor angiogenesis, lymphangiogenesis, and VM, it will be a candidate as effective treatment methods to improve not only cancer angiogenesis and metastasis but also cancer prognosis. In fact, accumulating evidence for therapeutic candidates associated with EVs indicate that several biomolecules in EVs have significant advantages for cancer diagnosis and prognosis . In addiNK, YY, SK, NA, and TO developed the concept of the review. NK, YY, and TO drafted the review. SK and NA corrected and reviewed the review. 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.


Diabetic foot ulcer (DFU) is a severe complication of diabetes and particularly susceptible to infection. DFU infection intervention efficacy is declining due to antimicrobial resistance and a systematic review of economic evaluations considering their economic feasibility is timely and required.To obtain and critically appraise all available full economic evaluations jointly considering costs and outcomes of infected DFUs.A literature search was conducted across MedLine, CINAHL, Scopus and Cochrane Database seeking evaluations published from inception to 2019 using specific key concepts. Eligibility criteria were defined to guide study selection. Articles were identified by screening of titles and abstracts, followed by a full-text review before inclusion. We identified 352 papers that report economic analysis of the costs and outcomes of interventions aimed at diabetic foot ulcer infections. Key characteristics of eligible economic evaluations were extracted, and their quality assessed against the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) checklist.542 records were screened and 39 full-texts assessed for eligibility. A total of 19 papers were included in the final analysis. All studies except one identified cost-saving or cost-effective interventions. The evaluations included in the final analysis were so heterogeneous that comparison of them was not possible. All studies were of “excellent”, “very good” or “good” quality when assessed against the CHEERS checklist.Consistent identification of cost-effective and cost-saving interventions may help to reduce the DFU healthcare burden. Future research should involve clinical implementation of interventions with parallel economic evaluation rather than model-based evaluations. Diabetic foot ulcers (DFU) are a common and severe complication of diabetes mellitus characterised by a deep tissue lesion.  Factors Not only is DFU a source of significant patient suffering, it also brings significant costs to the individual and healthcare system. The cost burden of DFU requires 6 days to 5.7 years of patient income to cover treatment cost with variation based on setting and treatment strategy.  The annuCost-effective DFU interventions have been identified in previous systematic reviews of economic evaluations, –13 howevA literature search was conducted using MedLine, CINAHL, Scopus and Cochrane Databases seeking articles published in English from inception to 2020. Terms including diabetic foot, economic evaluation and infection were used as Medical Subject Headings (MeSH) and textwords to capture the outcomes of interest. The MedLine search strategy, adapted for use in other databases, is presented in Studies were included if:they compared costs and outcomes in conjunction as part of a stand-alone economic evaluation or alongside a clinical trial or other study design types such as model-based economic evaluations,the study population was exclusively 18 years and over,the study population was diabetic with an infected foot ulcer,they were published in the English language in peer reviewed journals between inception and 2020.Studies were excluded if:costs and outcomes were not considered and/or compared,study population was not over the age of 18,they were theory papers, letters, editorials, reviews, theses, or dissertations and studies where full texts could not be obtained.This systematic review was conducted according to the Preferred Reporting Items of Systematic Reviews and Meta-Analyses (PRISMA) guidelines and the checklist is provided as supporting information . 14] Ar Ar14] ArKey characteristics of the economic evaluations were identified and extracted including study design and perspective, study population, intervention and comparator(s), time horizon and discount rate, methods or model used, costs included, reporting of costs, outcomes measuring health benefit and cost-effectiveness and overall economic evaluation result.Quality assessment of the reporting of identified studies was performed according to the 24-item Consolidated Health Economic Evaluation Reporting Standards (CHEERS) checklist.  Two reviPRISMA guidelines  were folStudy settings were diverse and accounted for South America, , 55 AustThe economic perspective taken by each study determines the cost and benefits included.  The sociA multitude of interventions were used to manage DFUs, typically antimicrobials or wound care strategies, reflecting components of the standard treatment of DFUs , 60 Tab. Most ecTime horizons should be specified and cover the provision of the intervention and tracking of costs and consequences/benefits. Ideally, they should reflect actual clinical practice. One-year , 54, 58 Four studies did not explicitly report the time horizon. , 51, 57 Discount rates allow economic evaluations to account for changes in the value of money over time. Four studies did not report a discount rate. , 49, 57 Eleven studies were CEAs, , 58, 59 Two studies did not use an economic model to simulate the impact of interventions on DFU. , 58 TiceThere was variation between cost inclusions in each study depending on setting, perspective and interventions investigated. Some studies kept a narrow scope of direct costs associated with the intervention , 56, 57 All evaluations except one concluded the intervention assessed was cost-effective or cost saving. This means all interventions provided more health benefit at a lower incremental cost most of the time. All CMAs showed the intervention achieved equal health benefit at lower costs versus the comparator.While the evaluations are incomparable due to heterogeneous methods and analyses, the intervention dominated the comparator in nine studies by providing greater health benefits at lower cost. , 54, 59 Adjuncts were dominant or cost-effective interventions in nine of ten papers. McKinnon et al.  and TiceThe reporting quality of each paper was assessed against the 24-item CHEERS checklist.  Studies Whilst the need for effective DFU interventions increases, few have been subject to economic evaluation. All interventions examined in these evaluations were cost-effective or cost-saving in a clinical situation involving DFU infection. Collectively, they suggested that short- and long-term implementation of such interventions could reduce the burden of DFU infection on healthcare systems while providing optimal patient management. Although the evaluations captured the standard care for DFUs and associated costs, other considerations were made on the issue. These included assessments of antibiotic efficacy, route and setting of administration and the overall strategies embodied in a variety of guidelines and recommendations.Seventeen evaluations were model-based and did not implement the intervention in clinical settings. –56, 59 TTogether all evaluations assessed at least one component of the DFU management strategy, with adjuncts assessed in most papers. Because the treatment of DFUs has multiple components including antimicrobials and standard wound care, it was to be expected that these economic evaluations would be too heterogeneous for comparison.Quality assessment of studies against the CHEERS checklist  found alThere are some limitations to our study. The results of this review rely exclusively upon studies published in English, which may not represent all research. Grey literature that is unpublished or published but non-commercially available was not searched due to time constraints. Full texts that could be obtained were limited to those accessible by the Flinders University library system. Although few studies were parallel evaluations to clinical trials, the majority of studies were model-based so the results relied heavily upon simulation of intervention effects rather than clinical application.In conclusion, economic evaluations have considered all aspects of DFU intervention, finding there is potential to select more cost-saving and cost-effective alternative to reduce the burden of DFU. Instead of model-based evaluations, future research should be directed toward actual implementation of interventions in clinical settings with economic evaluations in parallel.S1 Checklist(DOCX)Click here for additional data file.


Effective treatment options for patients with life-threatening neurological disorders are limited. To address this unmet need, high-impact translational research is essential for the advancement and development of novel therapeutic approaches in neurocritical care. “The Neurotherapeutics Symposium 2019—Neurological Emergencies” conference, held in Rochester, New York, in June 2019, was designed to accelerate translation of neurocritical care research via transdisciplinary team science and diversity enhancement. Diversity excellence in the neuroscience workforce brings innovative and creative perspectives, and team science broadens the scientific approach by incorporating views from multiple stakeholders. Both are essential components needed to address complex scientific questions. Under represented minorities and women were involved in the organization of the conference and accounted for 30–40% of speakers, moderators, and attendees. Participants represented a diverse group of stakeholders committed to translational research. Topics discussed at the conference included acute ischemic and hemorrhagic strokes, neurogenic respiratory dysregulation, seizures and status epilepticus, brain telemetry, neuroprognostication, disorders of consciousness, and multimodal monitoring. In these proceedings, we summarize the topics covered at the conference and suggest the groundwork for future high-yield research in neurologic emergencies. High-impact translational research is essential for the development of novel therapeutic approaches in neurocritical care. Although it is well known that diversity improves productivity in team science –4, thereAnother hallmark of the conference was the transdisciplinary participation of scientists from diverse backgrounds, including clinician-scientists, neuroscientists, bioengineers, rehabilitation experts, and trainees. Discussion topics within the conference included: early career development, funding strategies, diversity/team building, epilepsy and status epilepticus, neurotrauma, hypoxic pre-conditioning and neuroplasticity, cerebral edema and secondary brain injury, precision medicine in neurocritical care, neuroprognostication, advanced signal processing of neurophysiologic tests, disorders of consciousness, and neurogenic respiratory dysregulation.We believe that facilitating such collaborative research has the potential to contribute to the development of long-term networking resources that nurture diversity in team science. Targeted fostering has shown initial success in an example of a women science database . The talIn addition to reviewing the discussed content, we provide a blueprint to shape the future directions of neurocritical care research focused on novel therapeutics. An executive summary of the conference proceedings is shown in Table The complexities and multifaceted sequela of neurological emergencies require novel strategies for scientific progress and the development of ground breaking therapies with the potential to change outcomes and mitigate disability. In the current era of advanced subspecialties, such as neurocritical care, transdisciplinary teams are critical for health care team functioning , as wellTransdisciplinary teams include members with resourcefulness, strong delegation skills, diverse backgrounds, and trust in the process of multidisciplinary collaboration. The latter includes the establishment of psychological safety: the notion of standing up to speak on bold concepts, without fear of retaliation or negative repercussions. Diverse teams are also more likely to resolve complex problems , 18. HavSeveral studies have demonstrated that knowledge gaps and lack of exposure to racially and ethnically diverse groups, as well as sexual and gender minorities, may lead to barriers in access to clinical care for disadvantaged populations. This may stem from unconscious decisions influenced by the implicit biases of providers , 4, 28 ohttps://www.americanbrainfoundation.org/projects/) is the first neuroscience crowdfunding platform and has received nearly 150 donations and funded 8 projects (as of 2019). The National Institute of Health (NIH) Strategies to Innovate Emergency Care Research Network (SIREN) [Expanding funding mechanisms is essential to support diverse team science efforts . Under r (SIREN)  represen (SIREN) , 32 and  (SIREN) –35 have Further, efforts in harmonizing data collection in future trials of acute brain injuries, including curated multiple parametric and imaging data repositories, are urgently needed to facilitate subsequent data analyses and cohort discovery across populations.The following represents a summary of topics discussed during the conference. Due to the nature of its format as a live conference with interactive presentations and critical debates, this summary highlights the major points of discussion and perceived future directions of research proposed by the Neurotherapeutics Symposium 2019 attendees.Major advances in the treatment of ischemic stroke secondary to large-vessel occlusion have led to improved outcomes –35. PatiThe current patient selection criteria for endovascular thrombectomy (EVT)—particularly late-window—are relatively strict, which deprives a significant proportion of patients from targeted revascularization . DecisioBeyond reperfusion therapies, stroke-related therapeutics must expand to target white matter disease and cytotoxic edema pathways, and provide biologic biomarkers for patient individualized therapies , 47. PrePathways leading to cytotoxic edema after stroke represent another target for novel therapeutics with promising findings in pre-clinical and early clinical trials. Glyburide—an inhibitor of the ATP-sensitive potassium channel regulatory subunit sulfonylurea receptor 1—is an emerging therapy with favorable results on metrics of edema in the phase II RCT of hemispheric strokes . A Deepening the understanding of mechanisms underpinning secondary brain injury in cerebrovascular disease is another important step toward neurotherapeutics. Cortical spreading depolarization and depression—self-propagating waves of near complete breakdown of ion gradient homeostasis—is a potential mechanism of secondary injury in traumatic brain injury, acute ischemic stroke, intracerebral hemorrhage, and subarachnoid hemorrhage (SAH) often triggered by extravasation of blood, trauma or focal ischemia ; howeverThese parallel avenues of translational research seek to improve long-term outcomes after cerebrovascular diseases through novel neurotherapeutics. Harmonizing existing clinical databases and standardizing neuroimaging and neurophysiology interpretations for the purpose of inter-institutional collaborations sharing advanced expertise (such as the application of machine learning to big datasets) will facilitate these efforts .Chronic respiratory failure is a common and extremely disabling complication of motor neuron disease and cervical spinal cord injury , renderiIschemic preconditioning through acute intermittent hypoxia (AIH) is a promising therapy with potential to prevent respiratory deconditioning and enhance rehabilitation in chronic respiratory failure , 59. AIHThese translational and pilot human studies highlight the complexity of the biological mechanisms involved in neuroplasticity. The high variability in respiratory mechanics improvement depending on AIH methods, and the depth and duration of hypoxic events, support the hypothesis that respiratory motor neuron plasticity relies on balancing several and often competing pathways. These methods can have beneficial, but also pathologic consequences to patients; therefore, the refinement of AIH therapies in future studies is imperative. Biomarkers of short-term and long-term respiratory function will be fundamental to guide the delivery and monitoring of these therapies. Future studies should combine AIH strategies with pharmacological interventions, such as adenosine antagonism with caffeine or autoinflammatory modulation with corticosteroids—an approach with the potential to enhance the already promising results of AIH in motor neuron disease and spinal cord injury.Continuous electroencephalography (cEEG) is an integral part of the evaluation of critically ill patients with encephalopathy as nonconvulsive seizures are common in the critically ill and may negatively impact outcomes , 66. A cStatus epilepticus (SE) is a common neurologic emergency. Over the last decade, the role of pro-inflammatory mediators in seizure generation and propagation, and the search for novel therapeutic anti-inflammatory targets have been areas of intense research. Novel tailored immunotherapies targeting specific cytokines  have been shown to be effective in new-onset refractory status epilepticus (NORSE) and febrile-infection related epilepsy syndrome (FIRES) , 71. In Impairment in consciousness and cognition can occur in the setting of a primary neurologic disease , or as a consequence of a systemic insult . Invariably, an interplay of multiple factors concurrently contributes to the development of encephalopathy. As a result, predicting the short- and long-term consequences of impaired consciousness and cognition remains a challenge, in which the self-fulfilling prophecy bias of premature pessimistic outcome predictions plays an important role , 76. KeyOur understanding of the pathophysiology behind sepsis-associated encephalopathy (SAE) remains limited and is another area of much needed research in neurocritical care given the long-term disability associated with persistent cognitive deficits , 84. A cMultimodal monitoring in neurocritical care includes, but is not limited to, cEEG, intracranial pressure (ICP) monitoring, and measurement of cerebral blood flow surrogates, cerebral oxygenation, and cerebral microdialysis. The employment of MMM is mainly targeted to detect factors associated with the development of secondary brain injury, often as a result of bioenergetic failure, and monitor responses to targeted therapies. Various MMM subtypes aim to evaluate tissue function by assessing individual measures of metabolism, blood supply, or oxygenation. Further, MMM allows for the reliable detection of spreading depolarizations and their metabolic toll in cerebral tissue. For each parameter being assessed by MMM, there are several drawbacks that limit the expansion of this modality in clinical practice. Targeting individual values from MMM is unlikely to improve clinical outcomes in isolation. The assessment of average values may miss transient changes that, cumulatively, may result in a higher injury burden. The lack of integral data analysis precludes the incorporation of time in relation to the brain injury and the duration of secondary insults. Additionally, not accounting for individual resilience to insults, and the morbidity of certain therapies , challenges the ability of MMM to demonstrate a positive impact in clinical outcomes.The Neurotherapeutics Symposium 2019 included a transdisciplinary participation of scholars from diverse backgrounds, research interest and fields, and subspecialty focus. The conference highlighted the perceived clinically relevant research needs in various acute brain injuries and outlined future strategies on how to expand upon them while incorporating diversity and team science. We hope this format will also encourage continued mentorship and career building for early career investigators to help support and sustain a forward feeding pipeline of women and under represented minorities to advance translational research in neurologic emergencies. A focus on strong research networks, streamlined data sharing practices, and support for early investigators will increase the likelihood of important discoveries.


Youth Living with HIV/AIDS (YLWHIV) have a higher risk of developing immunodeficiency related illnesses including certain cancers than their general population counterparts of the same age. This narrative review of current available literature describes factors associated with pediatric access to oncological services, and the role economic strengthening could play in improving health outcomes for this vulnerable population. Findings suggest that both HIV-infected and -uninfected children living in low and middle-income countries struggle with access and adherence to cancer treatment and care. Cost of treatment is a major barrier to access and adherence. Asset-building savings programs may increase financial security and subsequently result in better health outcomes although they have not been utilized to improve access to cancer treatment. The HIV/AIDS pandemic continues to pose a significant threat to global public health . AccordiYouth 0–24 years) are greatly affected by the HIV pandemic. This can be explained by their diverse risk factors whereby children can acquire HIV from their infected mothers during birth and postnatally from breast feeding while 15 to 24-year-olds fall within a young adult category for which unsafe sex is the main risk factor for HIV contraction  years ar, 7. AddiAIDS-related illnesses contributing to mortality among people living with HIV are thought to develop as a result of HIV effects on patients' immune systems . ExampleHIV-associated cancer risk and survival among YLWHIV has changed over time with the advent of increased access to and uptake of antiretroviral therapy (ART) . Prior tThe WHO highlights the importance of early diagnosis for all pediatric cancers and access to treatment for children and adolescents with cancer for better survival . Proper This narrative review has the two primary objectives (1) examining access to oncological treatment services among children and (2) discussing the potential role of economic strengthening programs in improving health outcomes for YLWHIV who develop cancers.Articles were obtained through a comprehensive search of MEDLINE, CINAHL, CINAHL Plus, Ovid, PsycINFO, Academic Search Complete, Global Health, SocINDEX, SOCIndex with Full Text, and EBSCOhost's Clinical and Academic Collections using search terms in Out of more than 100 articles and internet sources identified from the search conducted over a period of ~4 months, material from 73 sources that provided information related to the papers' objectives was included. These articles include those obtained during the search (43) and supplemented sources (30) obtained from Google scholar searches and reference lists from included articles.While most YLWHIV live in SSA, cancer studies from this region are scarce. The number of YLWHIV who develop a malignancy in SSA is poorly defined and available data generally focuses on younger children (<16 years). HIV-related cancer surveillance is further hampered by lack of systematic collection of cancer diagnoses in HIV cohorts and absence of HIV status in available local cancer registries.Of an estimated 429,000 expected new cases of childhood cancer each year, almost 90% will occur in LMICs and over 80% of the global burden of pediatric cancer mortality is in LMIC , 23. A rStudies from lower income countries in the pre-ART era demonstrated a >40-fold higher risk of developing cancer in HIV-infected youth compared to their general population counterparts, with Kaposi sarcoma (KS), Non-Hodgkin's lymphoma and specifically Burkitt's lymphoma having the greatest relative risks –15. WhilYLWHIV generally have poor survival despite chemotherapy compared to uninfected peers, mainly due to patients' severe immunodeficiency , 30. HowAccess to pediatric oncology services is defined in terms of delay, waiting time, as well as treatment adherence and abandonment. Delay is further categorized as patient/parent delay, health care system delay, physician/health system delay, referral delay, diagnosis delay, treatment delay, lag time, and ultimately, total delay (defined as the time from onset of symptoms to initiation of therapy) , 34. TotSeveral barriers exist to accessing cancer treatment among children and consequently contribute to delay see . As chilLack of affordable local treatments options are amplified by long distances to treatment centers and transportation difficulties , 39. CliCaregiver education and profession also affect access to cancer treatment services. In a study in Argentina, retinoblastoma patients whose parents had an elementary education or lower had a greater risk of longer patient delay [odds ratio (OR) = 6.34] . In MexiIn a study conducted in Latin America, Asia and the Caribbean, household assets correlated with adherence to care . In UganA study in Israel, a high-income country, also observed a significant positive correlation between child's age at diagnosis and lag time whereby the lag time increased as the child's age increased . DiagnosCancer-related factors such as the cancer type, symptoms, stage, and tumor site/location also contribute to delays. Cancer type was significantly associated with delayed time to diagnosis especially when comparing different groups of cancers to leukemia, an effect which persisted even after adjusting for age, sex, and race . The timThe type of physician seen first (private vs. public), physician specialty (pediatrician vs. family physician), type of facility (private vs. public), health insurance, and the fear of visiting tertiary health care institutions also contributed to delays among children with suspected cancers . When thThis review could not identify studies that compared access to oncological services between HIV-positive and negative children. Yet, two U.S.-based studies focused on adults demonstrated that people living with HIV were less likely to receive cancer treatment and that the delay between diagnosis to treatment was also significantly longer than that of HIV negative individuals . The lacChildren with cancer in LMICs not only experience multiple barriers to treatment access, but they also have lower childhood cancer survival rates , 46. TheDespite improvement in outcomes for pediatric cancer patients in LMICs, many challenges persist. Interventions including targeted health education of guardians on the importance of early care for children's illnesses as well as advocacy and education of health workers to facilitate the identification of early warning signs of childhood cancer and foster timely referral and diagnosis are crucial for patient survival . Parent Similarly, regional collaborations between cancer centers have driven progress in pediatric access to treatment and survival . DevelopDeveloping sustainable and contextually relevant interventions, especially in resource-poor settings is imperative. While current interventions are vital and have resulted in increased access and government involvement in oncological care, socio-economic factors, and treatment-related costs play a significant role in the cancer-care pathway . Hence, This economic empowerment can be achieved using an asset-based model, which is an integrated approach for building human, social, and economic capital , 52. ThePeople living in poverty acquire personal financial resources either through savings or loans . HoweverAsset-based development through savings interventions has been used with success to improve quality of life. For example, the Suubi projects  in the Greater Masaka region in Uganda have employed an asset development approach in working with orphaned and vulnerable children in poverty -including YLWHIV, which among other components, involve setting up a Child Savings Account (CSA) , 59. CSACapacity building activities across all levels of health care systems are key to the care and treatment of children with cancer in LMICs—both with and without HIV infection. This approach requires collaborative work based on local stakeholder and government buy-in in order to set the priorities because they know best what they need and is feasible. Strengthening and expanding comprehensive pediatric cancer registries, especially in SSA will provide opportunities for research and innovative treatment approaches to improve survival. Collaboration between new and existing cancer and HIV registries for children and youth can further facilitate this process. Larger longitudinal studies are also needed to determine incidence of cancers in HIV-positive and HIV-negative children to better characterize the association between seropositivity and cancer.Established HIV-care systems and mechanisms at both local and regional levels should be utilized to provide cancer diagnosis and linkage to treatment services. Setting up centralized cancer treatment centers, partnering with established cancer units, establishing locally adapted protocols, and getting financial support from the government are all helpful in reducing treatment abandonment . CarefulResearch on diagnosis delays in childhood cancer is also important. There is also a need for more studies to investigate the potential impact of delays on prognosis outcomes. Future studies should study access to oncological services among YLWHIV, a high-risk group for pediatric cancer. Examining the impact of patient and provider/healthcare-system delays on disease severity and prognosis as well as the factors influencing these delays would inform effective policies and programs aimed at eliminating obstacles in the cancer-care pathway for youth with cancer.Finally, better strategies to reduce treatment abandonment and improve survival in childhood cancer in the developing world are still needed. Increasing awareness of childhood cancers and addressing structural and socio-economic factors impacting access to cancer treatment services can be considered as potential strategies. Moreover, complementing current structural interventions with the adoption of asset-based models to improve access and adherence to oncological treatment could be vital to pediatric cancer patients, especially YLWHIV.Pediatric cancers among youth are rare but serious life-threatening conditions. YLWHIV are particularly at a higher risk of developing and dying from cancer than their general population counterparts. Pediatric cancer patients have limited access to oncological services and health-system-related factors play a major role in delayed access to treatment. Failure to meet treatment costs is a major contributor to treatment abandonment. This paper discussed the potential role of socio-economic strengthening and provided evidence that asset-building savings programs can increase financial security and subsequently result in better health outcomes. Further exploration, development, and evaluation of interventions to address gaps and challenges to access to oncological services among YLWHIV and all children are crucial next steps.FS, OS, and KJ were principal investigators for the NIH funding. RK drafted the manuscript. OS, FS, and KJ reviewed and made substantive edits to the manuscript. 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.


The engraftment of human tumor tissues into immunodeficient host mice to generate patient-derived xenograft (PDX) models has become increasingly utilized for many types of cancers. By capturing the unique genomic and molecular properties of the parental tumor, PDX models enable analysis of patient-specific clinical responses. PDX models are an important platform to address the contribution of inter-tumoral heterogeneity to therapeutic sensitivity, tumor evolution, and the mechanisms of treatment resistance. With the increasingly important role played by targeted therapies in urological malignancies, the establishment of representative PDX models can contribute to improved facilitation and adoption of precision medicine. In this review of the evolving role of the PDX in urothelial cancer and kidney cancer, we discuss the essential elements of successful graft development, effective translational application, and future directions for clinical models. The increasingly recognized complexity of human cancers and the heterogeneity of malignant tumor cells have posed a major challenge to development of effective therapies in urologic oncology. Traditional cancer cell lines, the backbone of pre-clinical oncologic research, often fail to adequately address many of these conundrums, lacking the ability to faithfully recapitulate cancer treatment response and malignant progression. The engraftment of patient solid tumor tissues into immunodeficient host mice to generate patient-derived xenograft (PDX) models has been shown to provide a more valuable predictive paradigm in today’s era of personalized medicine and targeted therapies ,2. In prThe advent of PDX techniques date back to 1969, when a human colon cancer tumor was grafted into an athymic nude mouse . Howeverscid Il2rgtm1Wjl/SzJ strains . When utilized for research purposes, many studies have shown that these tumorgrafts retain the parental tumor histology, cellular structure, and genomic characteristics. At present, the cost of this process, variability in graft success rates, and the time required to establish the xenograft have been a barrier to the widespread implementation in clinical practice. While the oncology community remains optimistic that PDX models will one day serve as “mouse avatars,” providing patients and their clinicians with real-time information on tumor therapy response and tumor evolution, PDX models are not yet capable of serving as a decision-making tool. However, their role in the study of kidney and bladder cancers continues to expand, improving our understanding of disease biology.In what has been termed the age of “precision medicine,” there is increasingly recognized heterogeneity between tumors that contributes to variation in therapeutic responses. PDX models can play a role in assessing susceptibility to both traditional chemotherapeutic agents, as well as novel targeted treatments that exploit the biology of a patient’s unique tumor type. Creation of a xenograft model involves transfer of tumor fragments from a live patient’s primary tumor or metastatic site into immune compromised mouse strains, most frequently NOD (non-obese diabetic) scid (severe combined immunodeficient) or NOD.Cg-PrkdcKidney cancer and bladder cancer have a major impact on the American public, together accounting for more than 150,000 new cases annually, and comprising 8.8% of new cancer diagnoses in 2019. Moreover, there will be a total of 33,420 patients who succumb to these diseases, accounting for 5.5% of all cancer-related deaths [Urothelial carcinoma, which originates in the lining of the bladder and upper tract, is the sixth most common malignancy in the United States. However, it has an outsized impact on the US healthcare system, with the highest lifetime treatment cost per case of all cancers . Though Kidney cancer follows closely behind urothelial cancer as the eighth most common malignancy in the US. Again, like urothelial carcinoma, clinically localized stage I and II kidney cancers can be treated with extirpative management resulting in excellent survival. Unfortunately, treatment of locally advanced or systemic disease presents a significant challenge for clinicians, with a dismal 12% survival at 5 years in the metastatic setting . As in uIl2rg.Selection of immunocompromised mice for tumor engraftment plays an important role in the establishment of the PDX model. Researchers have developed successful models in four different types of mouse hosts: nude mice, scid mice, NOD (non-obese diabetic) scid mice, and mouse strains bearing targeted immunocompromising mutations in Nude mice are one of the earliest mouse strains established for human cancer research, thus named for their lack of fur. They are athymic, and their reduction in T cell production inhibits the adaptive immune response. However, their intact innate immunity can limit utility for human tumor grafting, particularly in less aggressive, slower growing tumor types, or in more immunogenic malignancies such as RCC and urothelial carcinoma . The sciThese issues with engraftment take-rate led to the development of more severely immunocompromised mice with impaired innate immunity, including NOD scid mice . The lacIl2rg led to the development of NSG (NOD.Cg-Prkdcscid/l2rgtm1Wjl), NOG (NODShi.Cg-Prkdcscid/l2rgtm1Sug), and NRG (NOD.Cg-Rag1tm1Mom/l2rgtm1Wjl) strains with targeted deficiencies in B cells, T cells, and NK cells. The deficiency in both the innate and adaptive immune systems allows for acceptably high take rates for human tissue engraftment [Il2rg-deficient mouse models (NSG/NOG/NRG), with reported rates of 0.7% in NSG and NOG mice [Finally, targeted mutations in raftment . Though NOG mice ,20,21,22Samples of primary and metastatic tumors sites that are resected surgically or biopsied are used for grafting. For RCC, sufficient tumor tissue can be taken at the time of radical or partial nephrectomy. After pathologic review, regions of the tumor specimen with viable (non-necrotic) malignant cells are brought to the lab for implantation. Unreliable tissue yields from tumor core biopsies make this a poor alternative. For bladder cancer, it is possible to establish grafts using tumor samples from both transurethral biopsy/resection specimens, as well as from radical or partial cystectomy specimens. Again, pathological confirmation by a genitourinary pathologist is a necessity, as chemotherapeutic treatment response or cautery artifact can affect tumor engraftment. Once specimens are removed from the patient, they should be placed in Roswell Park Memorial Institute (RPMI) medium or phosphate-buffered saline (PBS) solution and placed directly on ice. It is essential to minimize the time interval between excision of the tumor from the patient and engraftment in a mouse host in order to maximize tumor viability. In the case of renal cell carcinoma, when orthotopic engraftment into renal subcapsular space is planned, tissue slice grafts cut to 300 uM can be effectively used. Early models using this technique demonstrated ease of engraftment, and metastatic potential of the tumors. This approach also allows for generation of larger first-generation cohorts in the setting of limited tumor tissue .While PDX models are often maintained through serial passaging in mice, this can be costly and labor intensive. With limited resources, it is often necessary to freeze early generation xenografts for potential expansion at a later date. Cryopreservation with subsequent reanimation is an efficient and effective way to select tumors for PDX. Samples can be preserved at −80 °C in cryoprotectant 10% DMSO (dimethyl sulfoxide) media with 10% fetal bovine serum and RPMI solution. Alternatively, preservation can be done in a specialized cryopreservative , which has been shown to improve tumor reanimation engraftment efficiency ,25. Cryo3 should be implanted into this incision; bilateral engraftment can be achieved through the same incision if desired. Bilateral engraftment can be a more cost-effective way to increase tumor yield. Wound clips or surgical suture should be used to close the incision once graft is placed. Institutional standards should be used to monitor the mice after surgical implantation. Caliper measurement devices are used to measure tumor size in 3 dimensions at least bi-weekly, though more frequent measuring is necessary if tumor growth is rapid. Peri-procedural collaborative involvement of the institutional veterinary staff is essential to ensure appropriate care and optimal results. When indicated by the veterinarians, mice may be started on prophylactic amoxicillin or trimethoprim-sulfamethoxazole in their food, or enrofloxacin (Baytril) in drinking water to prevent graft or surgical site infection. In such cases, the antibiotic is typically continued until the host animal is euthanized.Once tumor tissue is adequately prepared for engraftment, host mice should be anesthetized using inhaled isoflurane. The area of fur overlying the anticipated incision should be shaved and sterilized using proper technique of betadine and alcohol. A small (<1 cm) skin incision is made in the flank or midback of the animal . A smallIt has been suggested that orthotopic grafting, namely implantation of tumor tissue into the renal subcapsular space for RCC or into the bladder wall or lumen for urothelial cancer, may better simulate the clinical disease course with development of distant metastases . While mTechnical challenges specific to the murine bladder have limited the development of orthotopic PDX models, with subcutaneous heterotopic implantation in the midback or flank reported in most published studies to date. However, various methods of orthotopic bladder PDX model creation have been described in the literature. One such method utilizes a small laparotomy incision to exteriorize the bladder, followed by drainage and direct injection of tumor cells into the bladder lumen . AlternaWhile evaluation of local tumor characteristics is important to guide therapeutic innovation, most patients eventually succumb to RCC and urothelial cancer due to metastatic spread. One of the major criticisms of PDX models, particularly heterotopic xenografts with subcutaneous implantation, is the lack of spontaneous metastases. Few metastatic models have been reported in bladder, though Tatum et al. described using BL0293, a patient-derived muscle-invasive bladder cancer PDX developed by Jackson Laboratory and University of California-Davis, as a reliable model for spontaneous and post-excisional metastatic disease to the liver. Notably, this model metastasized using a subcutaneous heterotopic implantation method . In theiA notable challenge in establishing a metastatic PDX model is non-invasive tracking of tumor spread. While cell-line derived xenografts can incorporate molecular markers such as luciferase to monitor for metastatic spread, this cannot be done with patient-derived tumor xenografts. Metastases can be identified on necropsy, but various imaging modalities allow for surveillance in survival studies. The primary tumor is monitored with calipers in the subcutaneous model, and orthotopic models can utilize ultrasound of the bladder or kidney, though image acquisition and interpretation are largely operator dependent. In their metastatic tumor model, Tatum et al. found that non-contrast T2-weighted MRI was the most effective way to accurately characterize metastatic burden in their mice .Generally, successful engraftment rates depend on a combination of host animal selection and tumor histology. High grade and advance-stage tumors are typically associated with an improved take-rate, owing to aggressive characteristics of the tumor cells. The same characteristics that allow a tumor to seed distant metastatic sites in humans appear to also allow for successful engraftment in mouse hosts. The faster growth rate of aggressive tumors also allows for more rapid expansion and serial passaging . In a stEngraftment rates in renal cell carcinoma can vary widely depending on the site of engraftment. Orthotopic renal subcapsular engraftment of tumor into the murine kidney has been shown to recapitulate tumor growth characteristics and metastatic potential ,33, but When the subcutaneous heterotopic approach is used to establish urothelial tumor PDX models, reasonably high take-rates can be expected. In a recent systematic review, Bernardo et al. looked at 12 studies using PDX models for urothelial carcinoma with an overall take rate of 41% , which cTo date, only six attempts at PDX creation from upper tract urothelial carcinoma (UTUC) have been reported with limited establishment and characterization . As the Xenograft models can only serve as a translational and preclinical platform to accurately predict tumor response to therapies if they preserve histology, gene expression, and tumor mutation profile . While tFollowing initial engraftment of the tumor into the host, human stroma is present with the malignant tumor cells, but with serial passages this is replaced by murine-derived stroma. When the endothelial cells, fibroblast, and other stromal elements known to affect cancer progression or inhibition are murine, there may be aberrant cross-reactivity with human-derived tumor cells. This has an impact on interactions with the tumor microenvironment, including alteration of paracrine signaling or murine cytokine reactivity. This limits the ability to examine therapies that target the tumor microenvironment in the PDX models and complicates the interpretation of drug response ,40.It is essential that the PDX model recapitulates not only the phenotypic or histological characteristics of the in-situ disease, but that the genomic and transcriptomic alterations are maintained in the PDX model after engraftment and multiple passages. Testing of the somatic mutational profile of the PDX for concordance with the original tissue can ensure the fidelity of the engrafted tumor and its response to targeted therapies. While earlier studies sought to confirm the preservation of tumor identity with immunohistochemical staining, developments in next-generation sequencing (NGS) have lowered costs and allow researchers to evaluate passaged tumors to ensure concordance with parental tumor identity. More rapid testing with NGS yield results within weeks. Pan et al. demonstrated through whole-exome and transcriptome sequencing that their PDX model of bladder cancer shared 92–97% of the genetic aberrations of the parental tumor. This allowed them to identify druggable targets, such as FGFR3, to serve as targets of molecularly guided therapies. Such high fidelity, with preservation of the parental tumor genetic aberrations through serial passages, is extremely important to ensure translational applicability of observed treatment responses .It has been shown that with serial passages, patient-derived cancer cell-lines will acquire DNA copy-number alterations that deviate from the parental tumor. This has been seen in RCC models as well ,43. SimiMolecular subtyping of both bladder cancer and RCC is an area of clinical research with important implications for PDX models. Transcriptional clustering by mRNA expression analysis in bladder cancer can be used to categorize tumors into subtypes with distinct phenotypes. Within these subtypes are luminal, basal/squamous, and neuronal-type tumors. While further validation is needed, there are potential predictive and prognostic implications of molecular subtyping that may eventually guide therapy decisions. As such, it is important that the different subtypes be reflected in PDX models, as there may be clinically significant differences in sensitivity to chemotherapy or targeted therapies. For example, luminal cancers that tend to be enriched in activating mutations in FGFR3 may better respond to FGFR3 inhibiting agents .Analogous to the molecular subtyping in bladder cancer, RCC expression profiles have been evaluated and may have similarly prognostic value. It is similarly imperative that RCC PDX models address differences in treatment responses seen in distinct subgroups identified using transcriptomic profiling . DiffereAn important limitation of genomic and transcriptomic sequencing in PDX models is the potential mixing of human and murine genetic content. To correct for this, reads mapping to human genes can be separated from reads mapping to mouse genes.The well-known limitations of human cancer cell lines have led researchers to search for preclinical cancer models that can reliably predict drug response in human trials. Alternatives to PDX include use of cell-line-derived xenografts, but these are known to lack heterogeneity of the disease in vivo. As discussed, they may acquire additional genetic alterations that lead phenotypic changes not reflected in the parental tumor . PDX mod1–3, mTOR, PDGFRα, MET, FGFR1–4, RET, KIT, and AXL have been targeted by established and emerging therapies [Traditional systemic chemotherapies have been notoriously ineffective against advanced renal cell carcinoma. As such, targeted therapies in RCC have become essential in the management of the disease when it has spread beyond the clinically localized setting. Numerous pathways including VEGFherapies . Variousherapies . In one herapies .In the treatment of urothelial cancers, platinum-based neoadjuvant chemotherapy (NAC) followed by radical cystectomy is the standard of care for muscle-invasive bladder cancer. While NAC has been associated with a pathologic response in 40% of patients with a complete response in up to 25%, there is significant overtreatment of non-responders . The expAt this time, approved therapy options targeting specific genes or biomarkers are limited in urothelial cancer. Erdafitinib, a tyrosine kinase inhibitor of fibroblast growth factor receptor (FGFR), was recently approved as the first targeted systemic therapy for metastatic urothelial cancer in patients with known FGFR2/3 mutations . While nIn the future, select patients with high-risk, clinically localized disease may be potential candidates for personalized PDX models. Tumor samples can be collected at the time of radical cystectomy  or nephrectomy (for RCC) to establish xenograft models. The PDX model can then be used to screen both standard therapies and drugs targeting specific molecular aberrations in the patient’s tumor. If the patient presents with disease recurrence, which typically takes several months, drugs with proven efficacy in the PDX models can be administered to the patient. Further studies demonstrating correlation between PDX response and patient parental tumor response are needed to validate such an approach. Furthermore, the cost of such as system has thus far prevented such integration into clinical practice. A more realistic approach is to generate PDX libraries with representative tumor subtypes and mutational variants. A focus on models that have been under-represented in cell lines would better reflecting the intertumoral heterogeneity of RCC and urothelial cancer. Once a library is established, specific targeted therapies can be screened for efficacy and resistance mechanisms.An understanding of the limitations of PDX models is essential for appropriate experimental design. When compared to traditional cancer cell lines, PDX models are costly and require substantial technical expertise. They can also be time consuming, with slow-growing tumors requiring months for successful engraftment.Immuno-oncology is an exciting and rapidly evolving field, particularly with the treatment of advanced bladder and kidney cancers. This therapeutic revolution has seen the recent approval of numerous immune checkpoint inhibitors . UnfortuIt is important for any researcher working with patient-derived xenografts or patient-derived cell lines to be aware of the potentially confounding effect that xenotropic murine retroviruses (XMRV) can have on growth parameters. There are reports of xenotropic murine leukemia viruses (MLVs) that are activated in immunodeficient mice with passage of human tissue. Though initially thought to play a causal role in the oncogenesis of prostate cancer, this has since been debunked. However, xenotropic viruses such as MLV, when activated, can induce spontaneous tumor formation in mice. It is suspected that there is relatively high prevalence of xenotropic viral infection in PDX models, with one study reporting detection of xenotropic endogenous murine leukemia virus in up to 53% of human breast cancer PDXs. Though the effect of XMRVs specifically on growth of kidney and urothelial tumors is unknown, PCR should be used to detect activated virus if there is any clinical suspicion. Periodic monitoring for XMRVs can also be done if there is concern for contamination. This also emphasizes the importance of sterile technique with tumor passaging to prevent widespread viral contamination of PDX models or cell lines in the lab ,84,85.The intertumoral heterogeneity that is seen in clinical practice is not reflected in genetically engineered mouse models, which tend to develop more homogeneous tumors. Tumor grafts are derived from a small area of the parental tumor, and thus fail to fully capture the heterogeneity of the entire tumor. Sampling multiple areas of the tumor for grafting can better capture the intra-tumoral heterogeneity that is known to be present in both bladder cancer and RCC, but this remains a limitation of any pre-clinical model.Building on experience with patient-derived cell lines, the utilization of patient-derived organoids (PDO) provides a less expensive alternative to solid tumor xenograft models. Organoids are three-dimensional tissue cultures and can be successfully established using human tumor cells. Lee et al. described 22 patient-derived bladder cancer organoid lines, and they successfully converted these lines into orthotopic xenografts using ultrasound-guided implantation. They demonstrated the ability to efficiently interconvert organoids and xenograft models in urothelial carcinoma . FurtherWhile no single in vivo system fully recapitulates the complexity of human cancers, combined pre-clinical approaches that include patient-derived xenografts are a growing and important component for research in urothelial and renal cell carcinomas. With the expanding number of both therapeutic drugs and better characterization of rational targets in urologic malignancies, the role for PDX models in elucidating susceptibilities and mechanisms of treatment resistance is expanding. Advancements in technical efficiency, improved understanding of limitations, and clinical validation of PDX models in bladder and renal malignancies will allow for improved translational application.


In recent years, several studies have elucidated the different signal transduction pathways involved in HM responses, identifying complementary genetic mechanisms conferring tolerance to plants. Although epigenetics has become more relevant in abiotic stress studies, epigenetic mechanisms underlying plant responses to HM stress remain poorly understood. This review describes the main epigenetic mechanisms related to crop responses during stress conditions, specifically, the molecular evidence showing how epigenetics is at the core of plant adaptation responses to HM ions. We highlight the epigenetic mechanisms that induce Al tolerance. Likewise, we analyze the pivotal relationship between epigenetic and genetic factors associated with HM tolerance. Finally, using rice as a study case, we performed a general analysis over previously whole-genome bisulfite-seq published data. Specific genes related to Al tolerance, measured in contrasting tolerant and susceptible rice varieties, exhibited differences in DNA methylation frequency. The differential methylation patterns could be associated with epigenetic regulation of rice responses to Al stress, highlighting the major role of epigenetics over specific abiotic stress responses.High concentrations of heavy metal (HM) ions impact agronomic staple crop production in acid soils (pH ≤ 5) due to their cytotoxic, genotoxic, and mutagenic effects. Among cytotoxic ions, the trivalent aluminum cation (Al Plants deal with multiple challenges to adapt to different environmental conditions given their sessile lifestyle. Abiotic stresses such as drought, salinity, extreme temperatures, nutrient deficiency, and heavy metal stress, represent some of the most limiting factors for plant growth .3 that belong to the Earth’s crust natural components. High concentrations of heavy metals can generate cytotoxic, genotoxic, and mutagenic effects in living organisms. Under physiological conditions, HMs can be divided into two groups: (i). Essential elements that are necessary for plant growth being structural blocks in proteins with an enzymatic function, such as iron (Fe), manganese (Mn), zinc (Zn), magnesium (Mg), molybdenum (Mo), and copper (Cu), and (ii). Non-essential elements like Cadmium (Cd), chromium (Cr), lead (Pb), aluminum (Al), and selenium (Se). While essential elements are necessary for plants in small amounts, high concentrations of both types of elements can lead to inhibition of plant growth and development (2+), calcium (Ca2+), phosphorus (P), sodium (Na+) and aluminum (Al3+) which in turn, affect plant physiological responses leading to crop yield losses for breeders and farmers  are elements with densities above 5g/cmelopment . Heavy m farmers .Acid soils represent nearly 30% of worldwide arable land, with 13% of staple crops cultivated in these areas. These types of soils classified as ultisols or oxisols are characterized by a pH lower than 5.5 . Al toxiStaple food crops such as maize, wheat, sorghum, and rice have been extensively studied to increase their Al tolerance . Among tImportant advances in elucidating the genetic mechanisms associated with HM tolerance and, especially, the molecular network involved in Al toxicity responses, have been reported in the last decade. Several studies on different crops have focused on genetic mapping to identify either quantitative trait loci (QTLs) or up/down-regulated genes associated with the response to Al stress . HoweverPlants have evolved different strategies to cope with HMs, diverging according to distinct factors as the plant species or the HMs exposure time and concentrations . These sHyperaccumulator plants have been important models to understand the possible mechanism by which plants have adapted to high HM concentrations, and to elucidate the putative genetic elements that could be involved in these processes . One rec3+ ions.Several studies have reported that tolerance or hyperaccumulation of HMs in plants is related to gene transcription modulation of metal chelators or transporters that favor exclusion or detoxification of the HMs . These g3+ ions in the rhizosphere (Triticum aestivum), sorghum (Sorghum bicolor), and barley (Hordeum vulgare) (One of the main strategies reported for Al exclusion is mediated by organic acid (OA) efflux from the root apex , a ubiquzosphere . The firvulgare) . Subsequvulgare) . Howevervulgare) .3+ ions through OA efflux; for instance, the MATE transporters OsFRDL2 and OsFRDL4, has shown a role in OA transport , have shown that transposable elements and repetitive sequences are the most heavily methylated DNA regions in the rice genome , which are the main CG methylases in charge of CG methylation; the plant specific enzymes chromomethyltransferases (CMTs), that are known to establish CHH and CHG methylation; and the domain rearranged methyltransferases (DRMs), that are involved in the maintenance of non-CG methylation and  and CHH . In cont and CHH .Abiotic stresses can generate a diverse range of phenotypes in plants, which are a consequence of complex molecular, biochemical, and physiological changes. Plants responses and adaptation to these stress conditions vary in different ways and at various levels, including short term physiological responses such as metabolic and gene expression changes, and long-term responses such as genetic and epigenetic genome modifications . The mecArabidopsis thaliana plants subjected to water-deficit regimes. When plants were irrigated, the H3K9ac modifications were rapidly eliminated, while H3K4me3 ones remained, indicating that the latter modification can be stably inherited through generations.DNA methylation states can be complemented by additional mechanisms such as histone modifications . AlthougA. thaliana was generated by exposure to drought, ABA, and salt stress, causing stress-responsive genes expression , which is sensitive to Cr, Ni, and Cd, and hemp (Cannabis sativa L.), which is partially tolerant to these HMs. The study found that in the absence of HM stress, the level of methylation of hemp roots was significantly higher than in clover. Similarly, Noccaea caerulescens plants (a Ni hyperaccumulator species) grown under high Ni concentrations were significantly hypermethylated at the genome level in comparison to A. thaliana Ni susceptible plants exposed to high Ni concentrations. These authors also showed that MET1, DRM2, and HDA8 genes, which are involved in DNA methylation and histone modification, were differentially expressed between N. caerulescens and A. thaliana. Hypermethylation has also been reported to act as a defense mechanism to counteract radiation genotoxic effect as shown by Pinus silvestris) adapted to survive high ionizing radiation, exhibited significantly hypermethylated loci compared to less adapted plants.A recent recurring question is whether there is a general pattern of DNA methylation related to HMs exposure in plants. Evidence from previous studies suggests that DNA methylation might play a role in the regulation of plant responses to HMs through at least two mechanisms . The firA second type of epigenetic response to HM stresses involves gene expression control . This reOryza sativa ssp japonica cv. Nipponbare). The authors found specific differentially methylated regions after Cd treatment, with patterns of methylation closely associated with transcriptional differences of stress response genes involved in metal transport, metabolic processes and transcriptional regulation. Likewise, some studies have shown the heritability and stability of HM stress-induced methylation changes  – metAFLP is a variation of the AFLP method. Nowadays it is poorly implemented given the emergence of genomic-scale methods. It is a cost-effective methodology that was used to elucidate methylation patterns in plants. The technique is able to detect global methylation marks throughout the studied genome. It is based on isoschizomers implementation to cut the DNA inside specific sites that display differential sensitivity to DNA methylation. A fragment comparison analysis reveals specific methylation polymorphisms. A major limitation is that it can only assess a small percentage of a global DNA methylation scenario. An important advantage is that these methods can be used for any species, even with limited or no information about their DNA sequence composition (MSAP (Methyl Sensitive Amplified Polymorphism) – This technique is a modification of the metAFLP technique described above. The protocol uses the EcoRI restriction enzyme in combination with the methylation-sensitive enzymes HpaII and MspI. These last isoschizomers recognize and cleave the same tetranucleotide sequence 5′-CCGG, but differ in the sensitivity to cytosine methylation. The method can differentiate among methylated, hemimethylated, or non-methylated sites. This technique was broadly implemented because of its cost-effective advantages, but one of its principal limitations is that it cannot specify the region or gene influenced by methylation (position . MSAP (Mhylation .CRED-RA (Coupled restriction enzyme digestion and random amplification) – Similar technique as the ones previously described. It is based on the use of restriction enzymes, specifically the isoschizomers HpaII and MspI implemented as Random Amplified Polymorphic DNA (RAPD) (A (RAPD) .HPLC (high-performance liquid chromatography) – There are several variants for this methodology but in general it involves the enzymatic hydrolysis of DNA to its deoxyribonucleotide components and subsequent separation and quantification of the nucleotides by high-performance liquid chromatography. The system gives highly reproducible results and, under suitable conditions, it is capable of measuring 5-methylcytosine levels even at low DNA concentrations. This method is implemented by comparing control samples versus treatments to evaluate genome-wide methylated cytosines. A major drawback is that the method is incapable of determining the sequence context of the methylated cytosine (cytosine .WGBS (Whole-genome bisulfite sequencing) – It is considered as the “gold standard” method in DNA methylation studies. This technique is based on whole-genome sequencing protocols, after bisulfite conversion of DNA. The bisulfite DNA treatment mediates the deamination of non-methylated cytosines into uracil, and these converted residues will be read as thymine, after subsequent high throughput sequence analysis. The main limitations are cost and bioinformatic analysis of NGS data, which can be overcome with reduced representation bisulfite sequencing (RRBS), where only a genome fraction is sequenced (equenced .Zea mays cv. RX9292), Al exposure can trigger DNA damage and cell death through a strong binding of Al ions to pectins and other structural components of the cell wall . AlthougNicotiana tabaccum cv Xan-thi nc). The study showed that Al stress promotes demethylation in the coding region of the glycerophosphodiesterase-like protein gene (NtGPDL) resulting in enhanced expression. NtGPDL belongs to the glycosylphosphatidylinositol-anchored protein (GAP) family linked to the extracellular matrix. Although the function of this gene is unclear, it seems to be involved in stress responses, including Al stress in tobacco , conferred enhanced Al tolerance to A. thaliana. This enzyme represents the main methyl group donor in plants and appears to play an important role in the epigenetic stress response. Overexpression of the AvSAMS1 resulted in changes both in DNA and histone H3 methylation after plant exposure to Al. More interestingly, there were differences in the demethylation and methylation patterns at different positions in the promoter and coding regions of this gene  insertion, located upstream of the coding region of the HvAACT1 gene. This gene is responsible for citrate efflux in roots, a mechanism involved in Al detoxification. The MRL insertion acted as a promoter and significantly enhanced HvAACT1 expression in Al-tolerant plants. This study showed that both the MRL insertion and gene expression, are due to demethylation processes, and are necessary for Al tolerance in barley. Additionally, transposon insertions close to genes have been proposed as a source of epialleles, and as a mechanism affecting the transcriptional regulation of specific genes . MoreoveEpigenetics has the potential to explain mechanistically, at least part of the molecular responses to different abiotic stresses, including HM toxicity . Althoug1). As a result, a group of 72 genes was kept, representing 10% of genes with the highest counts for methylated cytosines , while IR64 and Pokkali did not show differences in methylation  and ART1 (Loc_Os12g07280) as relevant in Al-related methylation. STAR1 encodes a nucleotide-binding domain that associates with STAR2, which encodes a transmembrane domain, to form a bacterial-type ABC transporter required for Al detoxification in roots  epigenetic marks are used as a mechanism to protect plants from possible DNA damage caused by metal ions through random DNA methylation along the genome, and (ii) epigenetic changes are used for the regulation of transposon and stress-responsive genes (Current knowledge of HM and Al tolerance in plants has been extensively documented with a direct focus on the physiological, and biochemical effects of these molecules, and their negative impacts on crop production. In rice, there is abundant information about genes and QTLs involved in Al tolerance in comparison with other staple cultivars such as barley or even the model plant ve genes .The studies carried out so far are evidence of putative epigenetic changes caused by HM exposure. However, it is necessary to evaluate the patterns of DNA methylation, as well as histone modifications occurring in precise genome regions to understand the possible epigenetic mechanisms underlying the regulation of the complex gene networks of Al tolerance responses. Likewise, there is a need for development of bioinformatics pipelines for epigenetic analyses. Future studies will be mandatory to evaluate the stability of the reported epigenetic changes through generations, given that epialleles can become permanent marks affecting genotypes and phenotypic responses. Finally, we report an overall greater abundance of methylated cytosines in an Al-tolerant rice variety, showing a contrasting methylation pattern related to differentially expressed Al responsive genes. This supports the hypothesis of DNA methylation as a fundamental key factor in the rice response to Al exposure.JG-F and CS performed the methylation analysis on Nipponbare, IR64, and Pokkali, and wrote and checked the manuscript. TG-H and MQ designed, edited, and checked the manuscript. 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.


Atherosclerosis is a chronic progressive vascular inflammation characterized by lipid deposition and plaque formation, for which vascular cell dysfunction and impaired immune responses are involved. Up to now, lipid-lowering drugs remain the main therapy for treating atherosclerosis; however, the surgical or interventional therapy is often applied, and yet, morbidity and mortality of such cardiovascular disease remain high worldwide. Over the past decades, an anti-inflammatory approach has become an important therapeutic target for dealing with atherosclerosis, as altered immune responses have been regarded as an essential player in the pathological process of vascular abnormality induced by hyperlipidemia. Interestingly, mesenchymal stem cells, one type of stem cells with the capabilities of self-renewal and multi-potential, have demonstrated their unique immunomodulatory function in the various pathological process, especially in atherosclerosis. While some controversies remain regarding their therapeutic efficacy and working mechanisms, our present review aims to summarize the current research progress on stem cell-based therapy, focusing on its immunomodulatory effects on the pathogenesis of atherosclerosis and how endothelial cells, smooth muscle cells, and other immune cells are regulated by MSC-based therapy. Atherosclerosis (AS) is now being regarded as an aging-associated chronic vascular inflammatory disease, which also involves the inflammatory and immune responses. Studies have shown that vascular endothelial cells (ECs), smooth muscle cells (SMCs), and immune cells have participated in and contributed to the atheroma formation, progression, and rupture at each stage of the atherosclerosis. While preventing the development or worsening of atherosclerotic lesion, such as the use of hyperlipidemia-lowering drugs, remains the major therapeutic strategy, which has significantly improved the prognosis of cardiovascular diseases, however, for patients at high risk or with detectable advanced plaque lesion, the progress of atherosclerotic plaque still has a high mortality rate . Thus, iIn addition to its inherent biological properties of stem cells, such as multi-potential differentiation and self-renewal capabilities, MSCs have become a hotspot in various research fields with their characteristics of rich sources, easy culture, and expansion . MSCs haEarly in 1960s, the transplantation of stem cells has been postulated to have a potential regenerative capability, which can be applied to treat the patients with cardiovascular diseases, especially for generating cardiomyocytes to compensate those lost in the event of MI . HoweverWhile BMSCs show their therapeutic effects on cardiac remodeling process through paracrine effects, studies have also indicated that immunomodulatory effects can also show their protective effects –14, such+ T cells and promoted the macrophage differentiation towards the M2 phenotype, thereby reducing the monocyte infiltration and inflammatory response [As reported, secretome from BMSCs dictates the bioactivity of immune cells, being of either pro- or anti-inflammatory property, which actually orchestrates inflammatory response during progress of cardiovascular disease. Considering AS as a chronic inflammation disease caused by a dysregulated immunity system, BMSC-based therapy might exert protective effects on pathological progression of atherogenesis Fig. . Growingresponse . Howeverresponse . A studyresponse . EVs derresponse . Interesresponse . For insresponse . Interesresponse . Both th+ adventitial progenitor cells showed enhanced capability of proliferation and migration and can differentiate into VSMCs and ECs contributing to the neointima formation in the setting of vascular injury [−/−deficient mice, research studies have found that several genes related to cell migration and matrix protein degradation displayed a significant alteration. Among them, a group of genes have a role in regulating progenitor stem cell migration; some genes were crucial for endothelial regeneration, and five genes were associated with leukocyte adhesion [Recent discoveries have uncovered the presence of a range of multipotent and lineage-restricted progenitor cells in the vascular adventitia, sharing high proliferative capacity and potential to re-generate ECs, VSMCs, and hematopoietic or mesenchymal cell progeny . Vascular injury . Using sadhesion , indicatadhesion . Howeveradhesion .Compared to allogeneic BMSCs derived from health donors, BMSCs from AS patients have significant differences in paracrine effects, either increasing the pro-atherogenic substances or decreasing athero-protective mediators . In factEndothelial cell dysfunction (ECD) is the primary cause of AS. Under the influence of AS high-risk factors such as lipid metabolism disorder and blood flow instability, the integrity of the vascular intima lining is interrupted, then continuous inflammatory stimulation and accumulating lipid deposition lead to the development of ECD. The dysregulated ECs mainly manifest the imbalance of endothelial nitric oxide synthase (eNOS) and NO production, leading to increased intracellular oxidative stress and a series of phenotypic changes in ECs such as inflammation phenotype and foam cell-like phenotype . On the −/−mice AS model [BMSCs may have the capability to regulate the endothelial progenitor cells (EPCs) homing to repair or regulate EC function. The number of EPCs is inversely related to cardiovascular risk, and patients with healthy vessels have a greater number of circulating EPCs . Though AS model , the vasAS model , 48. In AS model . A studyAS model . TransplAS model . HoweverAS model . In summ+ VSMCs have shown decreases in the expression levels of contractile phenotype marker proteins , and increases in secretory phenotype marker proteins , which were associated with their increased capability of migration and proliferation [Vascular smooth muscle cell phenotypic transition plays an important role in the progression and regression of AS. In the pathological environment, a series of alternations in gene expression of VSMCs have led to a transition from contractile phenotype (differentiated) to secretory state (dedifferentiated), manifesting as differences in proliferation, migration, phagocytosis, and secretion of inflammation substances and collagen fibers, all of which can affect the formation and progression of atherosclerotic plaque . In receferation .It is generally agreed that VSMCs has bidirectional roles in plaque formation. It has been shown that VSMCs secrete inflammatory substances and form foam-like cell in the early phase of plaque formation, whereas in the advanced stage of plaques, VSMCs secrete extracellular matrix proteins and collagen fibers to promote the formation of stable plaques . In factBesides, a study showed that SMC-specific ablation of TGF-β signaling in Apoe−/−mice fed western diet results in the reprogramming of VSMCs. These reprogrammed VSMCs have acquired MSC-like phenotype which can further transdifferentiate and generate osteoblasts, chondrocytes, adipocytes, and macrophages, resulting in the vascular abnormalities such as calcification and ossification of the aortic wall and aortic aneurysms . This phMacrophages is a key cell type that regulates the innate immune response during the development of AS. Bone marrow-derived monocytes recruit to the circulation and inflamed intima, which they differentiate into macrophages and dendritic cells (DCs) under the sustained stimulation of chronic inflammation, participating in a series of processes such as antigen presentation, lipid phagocytosis, secretion of inflammatory factors, formation of foam-like cells, and in the worst case becoming apoptotic and/or necrotic cells in the development of AS . It is w+T cell, which also provides a novel insight into the crosstalk between differentiated macrophage and T cells [After co-culture of monocytes with BMSCs, one study showed that the expression of macrophage surface protein arginase 1 (Arg1) was upregulated whereas inducible nitric oxide synthase 2 (iNOS) expression was downregulated; the secretion of inflammatory factors such as TNF-α was reduced in the cell culture supernatant where the anti-inflammatory factors such as IL-10 increased. Moreover, the foam cells decreased significantly after stimulation with oxidized LDL with the expression of lipid phagocytosis receptors Cd36 and Sra1 being downregulated, and the lipid transport receptors ATP binding cassette subfamily A member 1 (Abca1) and ATP binding cassette subfamily G member 1 (Abcg1) were upregulated , 62. Fur T cells . Of note T cells . Therefo+ T lymphocytes, while human atherosclerotic plaques have both CD4+ and CD8+ T lymphocytes. T lymphocytes can be divided into effector T lymphocytes (Teff cells) and Tregs according to their effects on AS. In the development of AS, phenotypic plasticity can be found to be gradually reduced, with the number of Treg cells reduced and Teff increased [T lymphocytes are the main cell type that regulates adaptive immune response in AS. Studies have shown that mouse atherosclerotic plaques are predominated by CD4ncreased . Therefoncreased . Currentncreased .Fig. 5T+ CD103+ within the plaque have the capability of modulating Treg differentiation [It is generally agreed that monocytes recruit to atherosclerotic plaque and differentiate into macrophages and DCs, being involved in the plaque formation. Interestingly, mature DCs that expressed CD11cntiation . A in vintiation .Besides, BMSCs exosomal miR-133 can downregulate the macrophage surface cholesterol efflux-related genes such as ABCA1 and ABCG1, resulting in an interrupted cholesterol efflux and inhibiting foam cell formation. Moreover, these changes are accompanied by a decrease in the number of athero-protective Tregs within the AS lesions, suggesting a potential link between immunometabolism in macrophages and induction of Tregs in the setting of AS pathological process . HoweverIn addition to the aforementioned endogenous progenitor cells, fibroblasts are the predominant resident population of the adventitia and responsible for depositing abundant collagen fibrils around vessels . Though Emerging evidence have shown that the spectrum of the key components within the secretome from BMSCs is mainly determined by the status of BMSCs which is closely related to their microenvironment. Pathological abnormalities such as calcifications and ossifications can be observed in nearly half of the MI mice receiving BMSCs therapy compared with those with purified hematopoietic progenitor cells, indicating potential deleterious effects of BMSC-based therapy . Even thThough the crucial role of BMSCs paracrine function has been increasingly recognized, it remains not fully understood how and to what extent the transplanted BMSCs can transit from circulation to the targeted lesions with an impaired vascular wall, as this is actually an initial step, yet a key step, for BMSC-based therapies in atherosclerotic plaque. Mobilization of BMSCs can be mediated by chemokines and inflammatory cytokines, either produced by inflammation cells or other activated BMSCs . Among tIn addition to the aforementioned findings, BMSCs derived from elderly subjects or patients with peripheral vascular disease showed a decrease in immunomodulation capabilities compared to BMSCs isolated from healthy donors, indicating that the different sources of BMSCs can influence the therapeutic effects of BMSCs. One of the advantages of MSCs is their rich origins, as MSCs can be isolated from various tissues, such as adipocytes, skin, bone marrow, placenta, and umbilical cord. Recent reports have found that MSCs derived from different sources exhibit distinct characteristics in their morphology, the success rate of isolation, colony formation frequency, maintenance period in cell culture, proliferation, and multiple differentiation capacity as well as their immunomodulatory effects  Table 11. An in MSCs, especially the BMSCs obtained from healthy donors, hold great potential for treating patients with AS for which the paracrine effects play the major roles; however, there also exist many factors which can potentially affect the biological functions of BMSCs, and these issues certainly need to be further clarified to facilitate their future clinical applications.Specifically, some essential aspects related to BMSC-based therapy warrant further investigations. First, due to the rich sources of MSCs, their different origin, probably different environment where the MSCs were isolated, will affect their immune- and inflammatory modulation capability. Data is lacking, especially side-by-side comparisons which should be made between MSCs obtained from these different sources when they are applied to treat patients with AS. Second, a different strategy would be carefully considered when BMSCs therapy was instituted for patients with stable or unstable AS, where the environment for transplanted BMSCs would be totally different, and the data of related basic research for this scenario is much lacking; therefore, much more carefully designed research needs to be carried out to better understand how the different inflammatory stages dictate the final result of BMSC-based therapy. Third, even though the paracrine effects have been regarded as the main underlying mechanism by which BMSCs mediate the inflammatory or immunomodulation, the specific molecule, such as peptides, small non-coding RNA, or other biological active products, should be identified, which would provide a novel therapeutic approach. Lastly, almost no data is available regarding whether and how the transplanted BMSCs are actually recruited to the targeted AS plaque; the detailed mechanism for BMSCs homing should be elucidated which would enhance BMSC-based therapeutic efficacy. Alternatively, the endogenous MSCs can also be re-activated by these fresh energized BMSCs from healthy donors which is probably another interesting mechanism involved in BMSCs therapy. The answers for all the research questions would certainly elucidate some fundamental scientific questions in this important research area and pave the way for wide clinical application for BMSCs therapy in treating patients with AS.