Social determinants of health (SDoH), which encompass the economic, social, environmental, and psychosocial factors that influence health, play a significant role in the development of cardiovascular disease (CVD) risk factors as well as CVD morbidity and mortality. The COVID-19 pandemic and the current social justice movement sparked by the death of George Floyd have laid bare long-existing health inequities in our society driven by SDoH. Despite a recent focus on these structural drivers of health disparities, the impact of SDoH on cardiovascular health and CVD outcomes remains understudied and incompletely understood. To further investigate the mechanisms connecting SDoH and CVD, and ultimately design targeted and effective interventions, it is important to foster interdisciplinary efforts that incorporate translational, epidemiological, and clinical research in examining SDoH-CVD relationships. This review aims to facilitate research coordination and intervention development by providing an evidence-based framework for SDoH rooted in the lived experiences of marginalized populations. Our framework highlights critical structural/socioeconomic, environmental, and psychosocial factors most strongly associated with CVD and explores several of the underlying biologic mechanisms connecting SDoH to CVD pathogenesis, including excess stress hormones, inflammation, immune cell function, and cellular aging. We present landmark studies and recent findings about SDoH in our framework, with careful consideration of the constructs and measures utilized. Finally, we provide a roadmap for future SDoH research focused on individual, clinical, and policy approaches directed towards developing multilevel community-engaged interventions to promote cardiovascular health. Many have clear and significant impacts on cardiovascular health and disease (CVD) outcomes for populations globally.8 These events highlight an urgent need to better operationalize structural SDoH components within both clinical and policy interventions for CVD risk reduction to reduce health disparities.Recent events such as the death of George Floyd, the subsequent protests that ensued, and significant health disparities highlighted by the COVID-19 pandemic have all underscored the role of SDoH in CVD outcomes. Specifically, racial and ethnic minorities have had the highest mortality rates in the pandemic, in particular before the availability of vaccines, due to structural racism and the disproportionate health effects of discriminatory policies and disparities in employment, housing, education, and health care access.10 Described as the biology of adversity, these biologic sequelae of structural inequalities, long-standing discrimination, and adverse social and environmental conditions require targeted translational and basic research to (1) identify key biologic markers associated with SDoH that may serve as effective CVD risk prediction tools and (2) develop targets for tailored and personalized interventions for those at highest risk for poor CVD outcomes.9 In this review, we provide a framework for the role of SDoH in CVD development and summarize biologic mechanisms that may associate these SDoH to CVD pathogenesis. Furthermore, we examine recent studies focused on the integration of SDoH into clinical care and cardiovascular health interventions.There has also been recent interest in the mechanisms by which SDoH influence biologic pathways involved in CVD development.11 For instance, both the Centers for Disease Control and Prevention and the World Health Organization define SDoH as the conditions in the environments where people are born, live, learn, work, play, worship, and age that affect a wide range of health, functioning, and quality of life outcomes and risks.13 While extensive, both frameworks fail to highlight key social processes, such as stigmatization, discrimination, and marginalization, that facilitate social exclusion and the isolation of vulnerable populations  individuals and others communities, and persons with low socioeconomic status [SES]).14 We argue that to work towards health equity, a SDoH framework should more critically focus on those most impacted by disparities given the disproportionate cardiovascular health effects of SDoH on these at-risk populations.The lack of clarity on the mechanisms by which SDoH affect cardiovascular outcomes may be partially due to a limited focus on the lived experiences of vulnerable populations within existing SDoH frameworks.14 emphasizes the social position of vulnerable groups to highlight their lived experiences and perceptions that develop upon interaction with social and built environments. Structural and intermediary determinants constitute the 2 domains of the framework. Within the structural domain, sociopolitical and economic context  define access to, and the quality of health care and education, SES, or neighborhood environment, as well as exposure to structural racism and discrimination. These high-level health equity determinants influence the intermediary determinants, including social and community context , which ultimately define social risk through housing instability, food insecurity, financial strain, or limited transportation.15 This framework disentangles structural health equity determinants into key constructs which allows a focus on their roles in the lived personal experiences of vulnerable populations, which may include implicit bias, everyday discrimination or stigma. This framework also allows for the exploration of the intersectional effects of broader sociopolitical, cultural and economic factors and intermediary health determinants on social position. Eventually, adverse SDoH in this framework can chronically stress the biology of disadvantaged groups, impact cardiovascular health factors, and promote disparities in CVD outcomes.Therefore, our health equity-focused framework Figure , revised16 Understanding these key constructs is also critical to address the knowledge gaps in how SDoH constructs with limited or no available longitudinal data impact cardiovascular health of marginalized groups. While Figure The constructs from the framework highlighted in this review were chosen based on evidence from longitudinal studies linking each to cardiovascular risk factors and CVD, as described in the following sections and outlined in Tables 18 Socioeconomic factors and, subsequently, other SDoH are influenced by structural determinants of health, or policies and systems put in place that dictate access to these resources. The links between structural racism and health inequities have been well documented.20 One way structural racism influences cardiovascular health of Black populations is through racial residential segregation.21 The current state of residential segregation is, in part, the result of a history of redlining, where areas of Black populations were flagged as hazardous investments by the government-established Home Owner’s Loan Corporation following the Great Depression.18 Discriminatory zoning, mortgage discrimination, and restrictive covenants further exacerbated the situation.17 Although the practice of redlining ended in the late 1960s, many neighborhoods remain segregated due to a legacy of social divestment in neighborhood infrastructure which perpetuate a disadvantage for predominantly Black neighborhoods.22 Currently, residential segregation has been shown to reduce employment opportunities and economic status, restrict access to quality education, and increase levels of neighborhood violence, crime and poverty.20 As such, the current state of segregation perpetuates health disparities observed in populations with low SES. In addition to SES, segregation may contribute to the development of psychosocial and environmental determinants of health as detailed in the following sections. Furthermore, it is important to consider the effects of structural racism that began before residential segregation, given that county-specific legacy of slavery associates with present-day heart disease mortality and may be a manifestation of intergenerational trauma.23Structural racism refers to laws, policies, and practices that were borne out of a history of discrimination and are now embedded within economic, cultural, and societal norms.24 In a multi-ethnic US cohort, Black populations living in areas with higher Black segregation had a 12% higher risk of incident CVD, independent of individual SES, CVD risk factors, and neighborhood characteristics.21 Conversely, White populations had a 12% lower CVD risk, but this was attenuated after adjusting for neighborhood characteristics. While the attenuation suggests neighborhood poverty may be a strong factor linking segregation to cardiovascular health disparities, the association of higher incident CVD with segregation in Black populations despite adjustment for neighborhood characteristics implies there are factors outside of neighborhood poverty also affecting cardiovascular health in segregated Black populations. Future research should consider broader domains wherein structural racism may operate. When political participation, employment, educational attainment and judicial treatment at the state level were considered as measures of structural racism, Black individuals in high-structural racism states were more likely than Black individuals in low-structural racism states to report a past-year history of myocardial infarction after controlling for age, sex, education, household income, medical insurance, and state-level poverty.24 The authors note that more research is needed to expand on indicators of structural racism by including factors like bank lending practices, access to education, and racial profiling.Overall, structural racism associates with cardiovascular health.20 For Hispanics/Latinx nationally in the United States, segregation has paradoxically been shown to associate with improved self-rated health (SRH) among those who were foreign-born, although poor SRH was reported in segregated US-born Hispanics/Latinx.25 The findings from segregated foreign-born Hispanics/Latinx may reflect the health-promoting characteristics of Hispanic/Latinx immigrant communities such as social support and protection from discrimination. In contrast, the poorer SRH reported among segregated US-born Hispanics/Latinx supports the idea that not only segregation but also social isolation may contribute to poor SRH. In a similar study investigating segregation and self-reported health in metropolitan Black individuals, it was found that social isolation had stronger associations with poorer SRH than did segregation alone.26Additionally, structural discrimination and its impact on cardiovascular health may vary by race and ethnicity.27 More research is needed to understand the differential development of health disparities within the historical context of diverse tribal nations, especially considering American Indian and Alaska Natives have experienced structural discrimination across many geographic areas stemming from a long history of European colonization and exclusionary US policies.28 The importance of investigating subgroups when examining segregation or structural racism is again demonstrated in a study of Americans who self-identified as Black but were further categorized as African American or Black immigrants of immediate Caribbean descent (Afro-Caribbeans). Increased residential concentration of Afro-Caribbeans was correlated with improved SRH while increased residential concentration of African Americans was correlated with poor SRH.29It is also important to investigate how structural factors differentially affect the cardiovascular health of various subgroups within affected populations. In the same national study of Hispanics/Latinx, US-born Mexicans and Puerto Ricans were found to be most impacted by segregation compared with other Hispanic/Latinx groups for reasons unknown. Similar subgroup heterogeneity is found among the different American Indian and Alaska Native tribal nations. While significant cardiovascular health disparities exist among American Indian and Alaska Natives overall, there are notable variations in the prevalence of cardiovascular risk factors  and CVD mortality rates across different tribal nations.31 and structural discrimination of individuals with disabilities.32While we have focused on structural racism, it is also important to recognize that many other forms of structural discrimination exist that can similarly affect health, such as structural gender discrimination35 This effect persists throughout the life course, as childhood low SES has been found to promote CVD events in adulthood36; however, more work is needed to examine the early life SES relationship with CVD independent of adult SES.37 It is also important to examine how SES-CVD relationships can vary by regions within countries, particularly in middle-income countries where the SES-strata at highest CVD risk may differ in urban versus rural areas.38 In low-income countries, available epidemiological data has indicated a relationship between education and CVD events.36 Additional investigation on the role of SES in CVD in developing countries is needed especially given the rising hypertension, hyperlipidemia, prediabetes/diabetes, and overweight/obesity rates in these countries.39 These trends are likely a result of industrialization, urbanization, and the subsequent transitions in dietary patterns, all of which can have a bidirectional relationship with SES.Numerous investigations spanning several decades have found strong connections between individual-level socioeconomic factors as SDoH  and CVD outcomes, with more recent data suggesting that lower SES serves a source of chronic stress that promotes a proinflammatory state and atherogenesis.41 Health care access as measured by insurance status is a particularly important SDoH. The expansion of health insurance access with the Affordable Care Act demonstrated that expanded insurance coverage for low-income populations was associated with greater health care utilization, including improved access to primary and subspecialty care as well as increased access to prescription drugs.41 However, it is less clear how greater insurance access affects CVD care quality or long-term CVD outcomes.43 Recent data suggest that the Medicaid expansion with the Affordable Care Act was associated with improvements in markers of hypertension and diabetes in low-income populations.44 While SES is a widely studied and important factor in both access to health care and CVD outcomes, we must also examine the intersection of this factor with other barriers like structural racism. Structural racism not only further reinforces and perpetuates the observed health disparities we see with low SES but also contributes unique damaging effects independent of SES.19Access to quality education and health care are highlighted as structural determinants of health equity in Figure 45 Built environments offer opportunities for physical activity through the connectedness of streets, mixture of land use, large scale environmental characteristics, and transportation systems.46 Recent reviews have identified certain attributes of built environments that associate with CVD risks and outcomes.48 For instance, residential density, traffic safety, recreational facilities, and street connectivity were consistently associated with physical activity and body mass index, while high density traffic and residential proximity to roads (defined as closeness from home to major heavy traffic roads) were associated with incident coronary heart disease.48 In Table 49 Research has also demonstrated that neighborhood greenness or vegetation is protective for cardiovascular health, possibly due to promoting physical activity and social contact, decreasing stress, and mitigating pollution, noise, and heat exposure.51 Future longitudinal studies should examine which specific built environment attributes influence factors like obesity and CVD, and how physical activity may mediate these relationships.47 Moreover, additional data are needed on how cardiovascular health disparities and built environment factors interact with hazardous weather phenomenon linked to climate change.53Neighborhood built environment, defined as the physical design of neighborhoods, is a key SDoH that influences CVD disparities.63 Higher area deprivation index as a marker of lower neighborhood SES was recently associated with higher cardiovascular mortality for individuals postmyocardial infarction at or before age 50 65 results in a spatial mismatch, which is a key limitation of prior built and social environment studies.67 To address this limitation, researchers have begun using global positioning systems to capture the environmental attributes of individuals’ activity space.70 Future research should focus on the built environment attributes around global positioning systems-defined activity space linked to cardiovascular health outcomes in addition to static (home-centric) approaches. Future research should also consider addressing potential causal interference from selective daily mobility bias. For example, if exposure to community parks from global positioning systems data is found to be associated with decreased body mass index, the exposure itself may not be the causal factor, rather the association may also be explained by intentional visits to the parks.72Most built and social environment research has exclusively focused on the residential areas of individuals, investigating the relationships between environmental attributes around the home (self-reported and objectively measured) with CVD risk and outcomes.73 In our equity-focused SDoH framework, we include the food environment in the social and community context in which individuals live, considering the food environment’s relationship with lived personal experiences that influences dietary behaviors  and chronically activate the sympathetic nervous system (SNS). Recent studies demonstrating how the immune system responds to chronic stress provide insights into how SDoH can lead to CVD. Uncovering how adverse SDoH may affect key signaling pathways also aids in our interpretation of clinical trial data and enhances our ability to assess the efficacy of interventions via measurement of key biomarkers of adversity described in the following sections. Here, we detail innovative studies investigating how SDoH induce stress hormones, inflammation, and other cellular processes that may contribute to CVD risk.We have reviewed constructs of our framework and their associations with cardiovascular health; however, it is important to recognize that precise biologic mechanisms linking these stressors to CVD remain largely unknown. This biology of adversity is a critical component in understanding CVD development in marginalized communities. One promising avenue of investigation examines how constructs previously described may induce psychological stress  leads to increased production of proinflammatory cytokines like TNF-α (tumor necrosis factor α) or IL-6 (interleukin 6).123 Increased frequency of discrimination, decreased social support, social isolation, and depression have all been linked to acquired glucocorticoid receptor resistance.126 However, more work is needed to systematically examine the role of SDoH in the development of acquired glucocorticoid receptor resistance.Glucocorticoid receptor resistance results from chronic activation of the SNS and the hypothalamic-pituitary-adrenal axis127 Norepinephrine and epinephrine signal through ARs (adrenergic receptors), a class of G-protein–coupled receptors. One subtype, the β2-AR (β2-adrenergic receptor), acts to increase intracellular cAMP which activates PKA (protein kinase A) to phosphorylate the transcription factor CREB (cAMP response element-binding protein). Under normal conditions, this cAMP-mediated process actively suppresses proinflammatory NF-κB signaling.128 However, evidence suggests that long-term exposure to adverse SDoH, including lower SES and social isolation, promote a switch from classical to noncanonical activation of β2-AR signaling. Noncanonical activation then redirects the β2-AR from cAMP signaling towards the G-protein-independent ERK  1/2 and MAPK (mitogen-activated protein kinases) proinflammatory pathway.119Chronic activation of the SNS also stimulates the sympatho-adrenomedullary axis which results in increased catecholamines, including dopamine, norepinephrine, and epinephrine. Catecholamines function as neurotransmitters and hormones, and are known mainly for their regulation of the fight or flight response.129 However, additional studies are needed to examine the impact of dopamine receptor signaling on immune cell function and subsequent CVD risk in the setting of chronic psychosocial and environmental stressors that serve as SDoH.The relationship between SDoH and dopamine is less well studied. In both acute and chronic stress, dopamine secretion appears to vary in a corticotropin-releasing hormone-dependent manner.131 and environmental stressors.132 In terms of socioeconomic determinants of health, data from the US Framingham Offspring Study cohort showed that educational level was significantly associated with CRP (C-reactive protein), sICAM-1 (soluble intercellular adhesion molecule-1), and MCP-1 (monocyte chemoattractant protein 1) in fully adjusted models.133 Recently, a meta-analysis found that lower SES associated with increased CRP and IL-6.130 In another study, exposure to low SES in early life resulted in increased CRP levels in adulthood when compared with individuals raised with higher SES.134Elevated markers of inflammation have been closely associated with both psychosocial stress135 found that high levels of perceived stress associated with various plasma cytokine levels, including IL-6, TNF-α, and MIP-1α and MIP-1β (macrophage inflammatory protein 1α and 1β).135 Risky emotional family environment in childhood predicted higher levels of IL-2, IL-6, IFN-γ (interferon γ), and TNF-α in adulthood136; childhood trauma was also found to be associated with increased IL-6 in adulthood.137Psychosocial determinants of health like perceived stress, childhood adversity, and discrimination have also been linked to inflammatory cytokines. For instance, Casaletto et al140 In a multi-ethnic study, associations between lifetime discrimination and inflammation burden were significant in models that controlled for sociodemographics, health behaviors, and psychological factors. Furthermore, poor global sleep quality was found to mediate the association between lifetime discrimination and inflammation.141 Future studies could examine whether disturbances in early life sleep pattern predict cardiovascular risk factors and CVD outcomes across the life course.Discrimination specifically has been connected to inflammation in various studies.132 An increase in circulating IL-6 levels has especially been seen in aging adults residing in neighborhoods with racial segregation and increased poverty.142 In a recent study from our lab, we demonstrated that within a cohort of Black individuals, neighborhood deprivation index as a marker of neighborhood-level SES was associated with increased levels of TNF-α and IL-1β.143 We also determined that neighborhood deprivation was associated with trimethylamine N-oxide,143 a biomarker associated with the gut microbiome, cardiovascular risk factors, and CVD mortality.144 Interestingly, our data showed that the neighborhood deprivation index-to-trimethylamine N-oxide relationship was significantly mediated by the TNF-α and IL-1β response,143 suggesting a need for future work to examine how inflammation induced by adverse SDoH may relate to the gut microbiome and CVD risk. Ultimately, more work is needed to link neighborhood factors to inflammatory markers and immune cell function.With regard to environmental factors and inflammatory cytokines, individuals residing in neighborhoods with higher rates of crime, violence, decreased walkability, low levels of social cohesion, decreased access to health care, increased pollutant exposure, and food insecurity display higher serum levels of CRP, IL-6, and fibrinogen.145 within the bone marrow and spleen, and thus affect the distribution and function of immune cells. By using 18F-fluorodeoxyglucose positron emission tomography/computed tomography imaging, we and others have found that increased amygdalar activity, a marker of chronic stress-related neural activity, associates not only with subclinical CVD but also with heightened bone marrow and splenic activity.147 This highlights the importance of the neuro-hematopoietic-inflammatory axis in CVD development and progression , Schmitz et al153 determined that epigenetic aging is accelerated in individuals experiencing socioeconomic disadvantage. Similar associations with accelerated epigenetic aging can be found at neighborhood-level SES.154 Investigations that describe DNA methylation patterns among individuals living in disadvantaged neighborhoods have shown alteration of gene expression particularly among genes involved in inflammatory pathways.155It is also critical to understand how DNA or epigenetic modifications might emerge due to a lifetime of disadvantage, structural inequalities, and discrimination. Biologic effects of SDoH on CVD risk may be passed on intergenerationally or can be related to gene-environment interactions.156 which has been linked to various chronic diseases, including CVD.157 Using self-identified race,158 Black individuals display greater telomere length at birth when compared with White individuals; however, this difference diminishes over the lifetime due to an increased rate of telomere shortening among Black individuals.159 Inflammation and oxidative stress, which are promoted by chronic psychosocial and environmental stress exposure, are likely the main mechanisms that promote loss of telomere length.162 Thus, numerous psychosocial and environmental stressors have been connected to telomere length or telomerase activity, including neighborhood deprivation,163 neighborhood disadvantage,164 low SES and educational level,166 early life stress,167 lower social support,168 increased early life adversity,169 high hostility,170 anxiety,171 and racial discrimination.172 Moreover, the association between lower parental SES and shortened telomere length in the newborn166 further demonstrates that the biologic effects of SDoH can reach across generations and influence health even before direct exposure to stressors. Future work should focus on the mechanisms by which SDoH affect telomere length so that targeted interventions may not only prevent development and progression of telomere shortening but also block this intergenerational transmission of adversity.In addition to DNA methylation, another cellular aging marker used to determine the impact of SDoH on genes is telomere length,Overall, it is important for us, as clinicians and scientists, to address the relationship between SDoH and cardiovascular health at multiple levels of intervention. On a biologic level, translational studies should include SDoH measures, and intervention studies would benefit from investigating effects on key biomarkers of stress and adversity. Animal studies are also important to study the biology of adversity in a more controlled environment. Furthermore, we must complement biologic investigations with targeted population health interventions addressing SDoH constructs that are known contributors to biologic adversity.173 The National Academies of Sciences, Engineering, and Medicine proposed a framework for improving social care through awareness of patients’ social needs through screening, adjustment of care to patients’ individual contexts, connecting patients to community resources, enabling institutions to prioritize resources in line with patient needs, and promoting policies that expand social care resources.174 Additionally, the American Heart Association has suggested expanding SDoH education for cardiovascular health providers at all levels, improving tools using electronic health records to incorporate SDoH screening and referrals into clinical practice, and expanding SDoH interventions to address upstream determinants of CVD such as poverty, education, and health care coverage.175Although it is well-established that incorporating SDoH screening and interventions into chronic disease clinical care significantly improves patient outcomes, current guidelines largely exclude SDoH-informed approaches.176 social risk score assessments,178 and health behavior counseling179 on reducing blood pressure, CVD risk, and LDL (low-density lipoprotein) levels.180 For example, the RICH LIFE (Reducing Inequities in Care of Hypertension: Lifestyle Improvement for Everyone)176 intervention sought to reduce hypertension control disparities through a collaborative care model that involved nurse care managers to coordinate care for patients with comorbidities, whereas the WISEWOMAN 179 intervention introduced lifestyle counseling from bilingual community health workers to improve cardiovascular behaviors among Latina women. Other interventions have targeted systematic approaches to SDoH integration in clinical care, such as developing a modified version of the Framingham CVD Risk Score177 and adding measures of SDoH to improve the predictive accuracy of CVD risk models.178 Major opportunities for integrating SDoH into clinical cardiovascular care can be found in standardizing electronic health records-based tools for SDoH assessments, facilitating panel management to identify and direct outreach to high risk patients, and tailoring clinical decisions to address environmental factors like housing conditions and health literacy.182 Clinical guidelines for CVD management can be updated to incorporate SDoH-informed care practices. Moreover, clinical education can be revised to better inform health care providers about how to identify and address their patients’ social needs. Health care institutions can also strengthen partnerships with community-based organizations to support the availability of social needs resources.Previous studies demonstrated the success of efforts such as community health worker or patient navigator programs,183 community input about needs and priorities should play a major role in the process of developing informed interventions at the individual, community, and policy levels. Ultimately, these interventions should contribute to multilevel strategies addressing cardiovascular health based on the foundation of community consensus and partnership.184 Consultation of community members can also provide insight into protective factors that critically influence health behaviors and outcomes,185 including community capacity and empowerment.186Following a community-based participatory research approach,187Mixed methods approaches combining qualitative and quantitative data are also critical in cardiovascular intervention development that incorporate SDoH. Over a decade ago, the Office of Behavioral and Social Sciences Research of the National Institutes of Health commissioned leaders in the mixed methods field to develop guidance for investigators on the rigorous elements necessary to conduct mixed methods research. Mixed methods approaches included (1) focusing on research questions that call for real-life contextual understandings, (2) using and integrating both rigorous quantitative and qualitative methods to fully understand and characterize constructs, and (3) framing the investigation within philosophical and theoretical positions.188In SDoH research, it is important to recognize that focusing only on quantitative variables provides neither a holistic nor accurate portrait of the person, family, or community. Understanding biologic mechanisms of CVD is necessary but not sufficient in designing health behavior interventions and public health policy to address CVD and SDoH. There are factors in the Critical Framework of Social Determinants of Health Figure  and thusSDoH have a significant impact on CVD risk and outcomes, particularly among marginalized communities. To address health disparities and promote health equity, it is essential to understand the various facets of SDoH, including the structural health determinants, built, food, and social environments, as well as the socioeconomic and psychosocial determinants of health. In addition to improving the measurement of SDoH in cardiovascular research and care, there is opportunity to build interdisciplinary teams that further investigate relationships between SDoH and the biologic mechanisms by which these determinants affect CVD risk and outcomes. Moreover, SDoH screening should be integrated into clinical care delivery, encouraging clinicians to tailor care delivery to the social needs of their patients. Finally, future research on the SDoH-CVD relationships should incorporate mixed-method approaches to better understand how individual-lived experiences of marginalization and discrimination affect cardiovascular health outcomes and develop tailored interventions informed by a nuanced understanding of social and environmental influences on cardiovascular health.We acknowledge our partners in the Washington DC Cardiovascular Health and Obesity Collaborative community advisory board, study participants, and former students and fellows, without whom none of our work would have been possible. We also like to thank the National Institutes of Health (NIH) staff and clinical teams and our collaborators for their continuous support.The statements and contents expressed in this perspective are those of the authors and do not reflect the official position of the National Institutes of Health (NIH), DHHS, and the US Government. The Social Determinants of Obesity and Cardiovascular Risk Laboratory is funded by the Division of Intramural Research of the National Heart, Lung, and Blood Institute and Intramural Research Program of the National Institute on Minority Health and Health Disparities. The Translational, Biobehavioral and Health Disparities Branch is funded by the Intramural Research Program of the National Institutes of Health, Clinical Center. The Neighborhood Social and Geospatial Determinants of Health Disparities Laboratory is supported by the Intramural Research Program, National Institute on Minority Health and Health Disparities, National Institutes of Health and by the NIH Distinguished Scholars Program. The Translational Biobehavioral and Health Disparities Branch is supported by the National Institutes of Health, Clinical Center. This research was made possible through the NIH Medical Research Scholars Program, a public-private partnership supported jointly by the NIH and generous contributions to the Foundation for the NIH from the Doris Duke Charitable Foundation, Genentech, the American Association for Dental Research, the Colgate-Palmolive Company, Elsevier, alumni of student research programs, and other individual supporters via contributions to the Foundation for the National Institutes of Health.None.


Renal fibrosis is a complex disorder characterized by the destruction of kidney parenchyma. There is currently no cure for this devastating condition. Extracellular vesicles (EVs) are membranous vesicles released from cells in both physiological and diseased states. Given their fundamental role in transferring biomolecules to recipient cells and their ability to cross biological barriers, EVs have been widely investigated as potential cell-free therapeutic agents. In this review, we provide an overview of EVs, focusing on their functional role in renal fibrosis and signaling messengers responsible for EV-mediated crosstalk between various renal compartments. We explore recent findings regarding the renoprotective effect of EVs and their use as therapeutic agents in renal fibrosis. We also highlight advantages and future perspectives of the therapeutic applications of EVs in renal diseases. Renal fibrosis is the final manifestation of chronic kidney disease (CKD), characterized by progressive destruction of kidney parenchyma and the subsequent loss of renal function. Due to the complexity of this condition and the inability to establish a proper hierarchy of involved mechanisms during the development of renal fibrosis, there is currently no effective antifibrotic therapy in clinical use . Over thIn recent years, numerous studies have pointed to EVs as important players in various physiological and pathophysiological processes, including kidney diseases. Apart from being active players in various biological processes ,3, EVs hThe current review explores contemporary findings on the functional role of EVs in the pathogenesis of renal fibrosis and signaling messengers involved in EV-mediated crosstalk between various renal and extrarenal cell types. We summarize the current knowledge on the renoprotective role of EVs from diverse sources and their use as therapeutic agents in renal fibrosis, highlighting benefits and new perspectives of therapeutic applications of EVs in renal diseases.EVs are membrane-limited vesicles that cells release in order to transfer biomolecules to other cells and thus communicate with them . In the EVs encompass diverse populations of membrane-limited vesicles released by virtually all cells in the organism. The diversity of EVs refers to their biogenesis, composition, size, and role . EVs are currently classified into two main types based on their biogenesis: exosomes and microvesicles (MVs) . In a wi++ ions-dependent enzymes, although it has been shown that specific members of the ESCRT complex are also involved in this process [In contrast to exosomes, microvesicles are formed by direct outward budding of the plasma membrane. Reshaping of the membrane is accomplished by changing its lipid composition at the budding site and disassembly of the cytoskeleton by Ca process ,20,21.Even though modes of biogenesis define two main types of EVs, their other properties are not so distinctive. As for composition, EVs can carry all types of biomolecules  involved in a number of processes such as adhesion, metabolism, signal transduction, membrane fusion, organization and trafficking, etc., in addition to molecules involved in EVs biogenesis ,8. The cAll these biogenesis and sorting mechanisms give rise to subpopulations of EVs with partially overlapping cargo . Due to EVs also differ in size and shape. Although it is often stated that exosomes are generally smaller than microvesicles (30–150 nm vs. 100–1000 nm), their sizes in fact overlap and thus cannot be used as a distinctive criterion for the determination of the EV type . As for EVs have numerous roles in the body. They are important players in virtually all physiological processes such as placentation, embryonic development, immunity, coagulation, liver homeostasis, nervous system function, tissue repair, and kidney function ,29. EVs Aside from their fundamental roles in the organism, EVs can have valuable functions as either diagnostic or therapeutic agents, or the combination of both, known as theranostics tools. EVs reflect the physiological state of the cell they originate from, and they are readily available in biofluids, and as such, they can be used in novel diagnostic platforms ,34. ThisSpecific properties of EVs such as biocompatibility, biological barrier crossing, selective targeting, and the possibility to be modified make these vesicles a very promising basis for the development of novel therapies ,6. ImporRenal fibrosis is the final manifestation of chronic kidney disease (CKD). It is a complex and dynamic process characterized by an uncontrolled accumulation of extracellular matrix (ECM), leading to a loss of functional renal parenchyma and consequently end-stage renal disease (ESRD), a severe disorder that warrants kidney replacement therapy . The patAfter an initial insult such as high ambient glucose, protein overload, hypoxia, persistent infection, autoimmune reaction, or chemical insults, injured tubular epithelial cells (TECs) of the kidney display dramatic cellular and molecular rearrangements including cell cycle arrest ,62, partRegardless of the fact that a range of conditions such as glomerulonephritis, diabetes mellitus, high blood pressure, atherosclerosis, obstructive nephropathy, interstitial nephritis, and polycystic kidney disease can be the major causes of CKD, renal fibrosis is always a key pathway in the progression of nearly every type of chronic renal disease ,72. In sNumerous studies have supported the role of EVs in diverse renal diseases, yet the knowledge of their function in renal fibrosis remains limited. Proteomic analysis of urinary vesicles confirmed that EVs could originate from all segments of the nephron, including glomerular podocytes, proximal tubules, thick ascending limb of Henle, the distal convoluted tubule, and the collecting duct . Apart fHere, we describe the contribution of EVs to different aspects of renal fibrosis. We depict how released EVs regulate/influence the destiny of renal and extrarenal cell types and subsequently the outcome of kidney injury. In addition, wherever possible, we describe signaling messengers from EVs responsible for EV-mediated crosstalk between various renal cellular compartments in renal fibrotic disease.Kidney autochthonous cells principally encompass proximal tubular epithelial cells (PTECs), distal tubular epithelial cells (DTECs), podocytes, and mesangial cells. An expanding body of data points to the role of EVs in the transfer of information between resident kidney cells, which subsequently induces damage of recipient cells and the development of renal fibrosis. It has been demonstrated that injured renal tubular epithelial cells could promote neighboring renal TECs to undergo epithelial–mesenchymal transition by transferring microvesicles (MVs) ,77. NameIt has been demonstrated that crosstalk between podocytes and renal tubular cells has an important role in the development of renal fibrosis. Indeed, exposure of tubular epithelial cells to microparticles (MPs) isolated from cultured media of untreated differentiated human podocytes led to a significant pro-fibrotic response . PodocytVarious studies have pointed to EVs as important players in the process of fibroblast activation. Borges et al. demonstrated that hypoxic stress led to the secretion of exosomes from injured tubular epithelial cells, which subsequently activated kidney fibroblasts promoting their proliferation and extracellular matrix production . The autRecruitment and activation of inflammatory cells are of paramount importance for the processes of renal inflammation and fibrosis. Various stimuli can trigger activation of inflammatory cells during the process of kidney injury facilitating their infiltration and perpetuating the vicious cycle of tissue damage. Recent studies have shown that various neighboring or distant cells can communicate with inflammatory cells via extracellular vesicle release. Lv et al.  showed tIt has been reported that during the development of DKD, the predominant macrophage phenotype is M1. M1 macrophages secrete inflammatory factors with a cytotoxic effect to glomerular mesangial cells and epithelial cells, thereby promoting the process of renal fibrosis in DKD . Jia et An increasing body of evidence demonstrates a renoprotective effect of extracellular vesicles, especially those derived from mesenchymal stem  cells (MSCs). MSCs are multipotent stem cells, which can be isolated from different sources including bone marrow, umbilical cord, fat (adipose) tissue, or placenta (amniotic fluid) . MSCs caSeveral lines of evidence described the role for bone marrow mesenchymal stem cell (BM-MSC)-derived EVs as therapeutic agents in renal fibrosis. Namely, Wang et al.  demonstrApplication of umbilical cord MSC-derived EVs has also been shown as a successful strategy to combat renal fibrosis in various models of disease. For instance, exosomes from human umbilical cord MSCs (UC-MSC) have been proposed as novel nanomaterials for regenerative medicine . Ji et aBeneficial effects of extracellular vesicles derived from adipose tissue mesenchymal stem cells (AD-MSCs) on renal fibrosis have also been demonstrated in different models of disease. In a porcine model of metabolic syndrome and renal artery stenosis, a single intrarenal delivery of adipose tissue mesenchymal stem cell (AD-MSC)-derived EVs attenuated inflammation, oxidative stress, and renal fibrosis ,104. TheSeveral lines of evidence point to the role of EVs derived from a healthy renal tubular cell in the protection against renal fibrosis. Namely, Dominguez et al.  showed tGiven all the presented evidence regarding functional roles of EVs and their beneficial effects in various models of renal fibrosis, it is conceivable that a new therapeutic approach for treatment/prevention of renal fibrosis, based on the use of EVs as target and/or tools, could be designed. EVs are generally considered to have great therapeutic potential due to their unique set of properties. Namely, EVs are natural, biocompatible products; they carry multiple information and are able to cross many biological barriers. They can be targeted and modified to carry drugs and/or target molecules. Importantly, EVs can replace cell therapy, i.e., MSCs, since they can convey cell benefits while having a critical advantage over the use of live cells due to their inability to proliferate and cause tumors ,115,116.One approach in this new line of therapy would be designing a therapeutic that would interrupt EV-based communication between cells in the processes leading to renal fibrosis by blocking the production or uptake of EVs. Studies of the release and uptake of EVs have identified several molecules involved in these processes and several compounds that could inhibit them. EV release can be inhibited along their different pathways of biogenesis by affecting EV trafficking or lipid metabolism as comprehensively reviewed by Catalano and O’Driscoll . Thus, EAlternatively, EVs themselves could be applied as therapeutic agents B. As staAnother approach of EV-based therapy would be a modification of EVs aimed at increasing their efficacy B. Thus, The final approach encompasses a design of artificial EV-like vesicles, i.e., constructs consisting of a lipid-limiting membrane with targeting molecules on the surface of the vesicle and an active therapeutic cargo C 124,12. This apOne of the often-mentioned issues regarding the use of EVs as therapy for renal diseases refers to the inability of EVs to pass the slit diaphragm due to a bigger size of the EV than the slit pore size ,132. ThuExtracellular vesicles are essential emissaries of information between cells and important players in various physiological and pathophysiological processes. EVs can enter blood vessels and reach different parts of the body transferring bioactive molecules such as proteins, RNAs, and lipids to distant target cells. It has been demonstrated that EVs play essential roles in tubulointerstitial communication during renal fibrosis since injured kidney cells may transfer EVs carrying various molecules to recipient cells, hence actively participating in renal inflammation and fibrosis.One possible target of therapeutic EVs in renal fibrosis could be the renal tubular epithelial cell (TEC). After an injury, TECs display dramatic cellular and molecular drift that consequently leads to alterations of their epithelial secretome profile, making TECs important players in renal fibrosis. Injured TECs release EVs that can promote neighboring TECs to undergo EMT or activate fibroblasts and induce macrophage M1 polarization and their recruitment. EVs released from various MSCs and other cell sources have emerged as a powerful cell-free therapy in different models of renal fibrosis. MSC-derived EVs may suppress EMT of TECs, fibroblast activation, and myofibroblast transdifferentiation in vitro, shuttling different miRNAs and proteins. Furthermore, administration of MSC-derived EVs ameliorates tubular injury and interstitial fibrosis in different animal models of renal fibrosis.Targeting EVs directly to inhibit their pathogenic effect or take advantage of their potential regenerative roles could be some of the promising therapeutic strategies. On an odyssey toward new therapeutic approaches for renal fibrosis lay numerous issues to be resolved; nevertheless, both the nature of EVs and the widespread efforts to make EV-based therapies in different areas, render EVs a promising tool in designing a new generation of therapy for renal fibrosis.


Ewing sarcoma, a highly aggressive bone and soft-tissue cancer, is considered a prime example of the paradigms of a translocation-positive sarcoma: a genetically rather simple disease with a specific and neomorphic-potential therapeutic target, whose oncogenic role was irrefutably defined decades ago. This is a disease that by definition has micrometastatic disease at diagnosis and a dismal prognosis for patients with macrometastatic or recurrent disease. International collaborations have defined the current standard of care in prospective studies, delivering multiple cycles of systemic therapy combined with local treatment; both are associated with significant morbidity that may result in strong psychological and physical burden for survivors. Nevertheless, the combination of non-directed chemotherapeutics and ever-evolving local modalities nowadays achieve a realistic chance of cure for the majority of patients with Ewing sarcoma. In this review, we focus on the current standard of diagnosis and treatment while attempting to answer some of the most pressing questions in clinical practice. In addition, this review provides scientific answers to clinical phenomena and occasionally defines the resulting translational studies needed to overcome the hurdle of treatment-associated morbidities and, most importantly, non-survival. Althougr entity , which ar entity . For allLocal therapy in patients with EwS is highly individualized. Patients should have the opportunity to explore local treatment options as soon after diagnosis as possible and decisions about local therapy should be made in collaboration with patients and families . Expert The optimal approach for local control in patients with EwS is influenced by a multitude of factors, e.g., patient age, tumor site, size, and local extension, and remains a matter of discussion. Randomized studies comparing surgery and radiotherapy (RT) in general, and their timing and sequence in particular, have either been limited or not been performed ,101. StiThe risk of local recurrence has to be carefully weighed against the functional outcome and late effects of either modality. For the latter, late effects such as growth impairment and second malignancies following RT have to be balanced against periprosthetic infections using tumor endoprostheses or pseudarthrosis after biological reconstructions, bone healing difficulties, and fractures following surgery ,110,111.Definitive surgery typically follows an initial period of neoadjuvant chemotherapy, unless emergency surgical procedures are mandatory at diagnosis, e.g., in case of spinal cord compression. Importantly, patients should be referred to an experienced center for their operation. The timing of surgical local therapy is primarily dependent on the duration of neoadjuvant chemotherapy but is also determined by the availability of the necessary technical devices  and the most experienced interdisciplinary surgical and/or radiation therapist team ,113. ThuThe principles and techniques of surgical resection and reconstruction in primary malignant bone tumors have been defined by Enneking . The aimReconstructive surgical techniques should be applied wherever possible, but oncological control trumps limb preservation. The majority of patients with surgical local treatment requires bone reconstructive surgery. Biological reconstruction with bone grafts is one option, in particular in intercalary bone defects preserving the original joint. Techniques commonly used are vascularized fibula grafts, allografts or irradiated autografts. All of those excel at different advantages and disadvantages in terms of complication or reconstruction survival.Today, tumor endoprostheses are widely used for bone reconstruction, in which a joint replacement is necessary. Endoprosthetic replacement in growing children requires specific expertise due to their rare indication: the small anatomy of the immature patients, the loss of the epiphysis in metaphyseal tumors require specific individualized implants. Custom made implants with small volumes adapt to the small anatomy. Non-invasive growing implants provide the opportunity to replace sacrificed growth plates and are able to compensate length discrepancies . While rPrimary amputation is rarely needed in EwS due to both tumor shrinkage during neoadjuvant chemotherapy and the availability of definitive RT as an acceptable alternative to surgery. Ablative surgery should be restricted to patients with severe complications after surgery and in tumors in which limb salvage would compromise the necessary surgical margin, e.g., extended infiltration of the neuro/vascular bundle, in cases of tumor progression under neoadjuvant treatment or in very young patients below the age of 3–6 years with tumors of the lower extremity. Secondary amputation is mainly due to local recurrence or periprosthetic infection. Comparative assessments of the quality of life in patients undergoing amputation or limb salvage surgery have not revealed any differences in long term outcomes . RotatioRegardless of the surgical method chosen, it should be emphasized that the long-term functional outcome of surgical local therapy depends on lifelong physical and psychosocial rehabilitation . Functional deficits do not occur only in the area of the reconstruction site. Often, adjacent joints, the contralateral extremity, or the spine  cause more problems than the surgical site .The surgical margin status is a reliable indicator of tumor left in the patient . An adeqIn clinical practice, sufficient margins are carefully weighed against the functional outcome following surgery, in particular for pelvic EwS. For soft-tissue components, which usually shrink after neoadjuvant treatment, a downstaging strategy is discussed with the preoperative radiological detectable tumor guiding the extent of resection, while the same rules of intra-compartment margins apply. This strategy potentially omits areas of formal tumor seeding . Noteworthy, these are non-evidenced based recommendations. So far, several studies have attempted to quantify the margins of resection, but no consensus has been reached, and debates are ongoing ,124. WheAn open biopsy channel must be completely included in the surgical specimen after open biopsy , while tHistopathological response has a major impact on local control rates in European studies . While dNeoadjuvant chemotherapy alone or in combination with preoperative RT often results in a substantial reduction in tumor volume, especially of the extraosseous component, and facilitates or even enables adequate limb-sparing surgery. Persistent extraosseous tumor growth after neoadjuvant systemic treatment in patients with non-sacral EwS tumors undergoing surgical treatment might be an important indicator of reduced overall survival probability . In patiPathological fractures are indicative of a large and highly aggressive tumor and the hematoma associated with the fracture could possibly result in spreading of the tumor into the surrounding soft tissues . In EwS,The standard of local care in patients with resectable tumors of the extremities has been considered definitive surgery which in most cases can be limb sparing . Wide reData on regional chemotherapy by perfusion of EwS of the extremities are lacking.per se does not necessarily improve survival but may significantly limit the patient’s quality of life. Still, in the clinical care of pelvic and sacral EwS, the associated side effects of the local therapy modalities must be weighed against each other: While surgical intervention in small pelvic tumors, depending on their location, can mutilate and may lead to significantly reduced quality of life , RT carries the risk of osteoarthritis. In this context, published outcomes for photon beam radiation may be surpassed by proton beam radiation in the future (please see section “Modern RT strategies and techniques”) [Historically, primary pelvic EwS had the least favorable prognosis compared with all other sites with higher rates of local relapse and reduced survival ,136, butniques”)   and far less often from the soft tissue of the chest wall, the lung or the mediastinum . The aimper se if the tumor is deemed resectable. Tumor resectability needs careful preoperative multidisciplinary evaluation including orthopedic spine surgeons or neurosurgeons and thoracic surgeons. Noteworthy, the type of spinal reconstruction can affect the choice of RT treatment modality [In rare cases, EwS of the chest wall extends to the spine. Intraspinal infiltration is not a contraindication for surgery with curative intent modality . Figure Chest wall reconstruction may be required to cover chest wall defects and preserve respiratory mechanics. Depending on the size and location of the chest wall defect, it is usually performed combining synthetic materials and/or pediculated/free musculo-(cutaneous) flaps. Primary soft-tissue closure is, however, possible in most cases . While pPrimary pulmonary and mediastinal EwS are very rare. They are described mostly as case reports or appear as single entity in larger studies ,158. In Surgery for primary mediastinal EwS should lead to complete tumor resection with free resection margins. Due to close anatomical relation to unresectable anatomical structures  EwS of the mediastinum may not always be resectable ,154,156.EwS is a rare disease and knowledge about diagnosis and treatment strategies determine patients’ survival and it is strongly recommended to refer patients with suspicion of a soft-tissue tumor to an experienced center. Perhaps more than for most cancers, the pathway to appropriate EwS treatment may already have been declared by events that have taken place prior to referral. For example, the type of biopsy that may have already been performed, or a prior inappropriate excision may jeopardize the form and outcome of local treatment thereafter ,159. PatIn general, tumors which cannot be completely resected must be considered inoperable, since EwS patients do not benefit from debulking procedures . Tumors Local therapy of involved sites is important for patients with primary, disseminated, multifocal EwS and should complement systemic treatment whenever possible. Outcome with local treatment of both primary tumor and extrapulmonary metastases is superior to results with local treatment of either the primary tumor or extrapulmonary metastases . In dissLocal control including surgery and/or RT may play an important role for some patients with relapsed EwS depending on the timing of relapse, location of relapse and sensitivity to chemotherapy. For localized relapsed EwS patients, local control strategies with either surgery or RT may improve outcomes and should strongly be considered when feasible ,161, butThe radiosensitivity of EwS has been recognized since its first description by James Ewing . RT as aReasons for preoperative RT include tumor progression or anticipated marginal or intralesional resection. The AEWS1031 Ewing protocol preferentially recommends preoperative RT for apparently resectable tumors in selected sites such as the pelvis, chest wall, and axially tumors where there is a higher risk of R+ resection. Postoperative RT is indicated in case of intralesional (including intraoperative spill) or marginal surgery (≥R1 resection). In European protocols, postoperative RT is also used in case of poor histological response (≥10% of viable tumor cells within resected tissue), regardless of surgical margins ,107,168.In addition to achieving sufficient local tumor control, long-term tolerability and minimization of adverse effects of RT are major goals of modern RT techniques. Children are particularly vulnerable to radiation-induced late toxicities and secondary malignancies due to their immature tissue. Nowadays, smaller treatment area and consequently minimized dose burden to normal tissue are the results of individually optimized and very high precision RT application . The optWith regard to the optimal fractionation regimen, hyperfractionated, accelerated regimens with, e.g., 1.2 Gray (Gy) per fraction twice daily, have been shown to have fewer long-term side effects and better functional outcomes for extremity or pelvic EwS ,181. HowCombined strategies of surgery and RT yield to more favorable outcomes in local control than either definitive RT or surgery alone ,183,184.Therapy for EwS is based on national and international study protocols (please see section “Systemic therapy”). Total doses between 45 and 60 Gy are typically prescribed for primary tumor sites with a 2 cm security margin . FractioAlthough the local control rate in the adjuvant setting has been around 85–90% in most series ,153,186,Both initial and residual tumor area as well as areas of potential microscopic extent have to be covered by the target volume of RT. For tailoring target volume concepts, it is crucial to restrict higher dose levels to areas carrying the highest risk for failure . CurrentFor disseminated EwS, RT can be important component of multimodality therapy. In addition to the primary tumor, all extrapulmonary sites should be attempted to receive small volume irradiation of about 45 Gy. If this policy would exceed the volume of 30% of the patient’s bone marrow, local field RT should be restricted to the most relevant areas in order to avoid extensive bone marrow suppression. Autologous bone marrow transplantation may be considered after extended irradiation. Particularly for bone metastases in EwS, RT was effective to achieve local control . With wiWhole-lung irradiation (WLI) is indicated if exclusive pulmonary metastases are present at the time of diagnosis, even when complete remission was obtained with chemotherapy . Both luIn the relapsed setting, RT may be used similar to first-line strategies for EwS patients who relapsed ≥two years after the beginning of first-line therapy and/or who present with exclusive pulmonary metastases ,191. In Only a minority of patients showed significant pulmonary function abnormality after WLI . The risHistorically, the potentiation of radiation pneumonitis by dactinomycin/actinomycin D has been well described both in vitro and in vivo ,198,199,Local and disseminated recurrences in EwS typically have a dismal prognosis. Nevertheless, local treatment may provide medium-term local control, particularly in cases of preceding local failure. However, re-RT has to be evaluated carefully as previously applied doses and burden to important organs at risk have to be taken into account—at least with regard to short and medium toxicity. Currently, no standards for palliative RT in EwS are defined. Some studies on EwS include palliative RT in case of symptoms. One study revealed that RT is an effective treatment for metastatic EwS in palliative settings and offers a chance to provide symptom relief . Pain, fPrior to the 1970s, EwS tumors were treated solely with surgery and/or RT with nearly all patients eventually developing either primary or distant relapse ,204. Folvincristine and cyclophosphamide, and vincristine, actinomycin-D, cyclophosphamide and doxorubicin (adriamycin) (VACA), both of which demonstrated improved survival compared to historical controls [2 and carried an unacceptable degree of cardiac toxicity. Finally, the IESS-1 randomized trial in the 1970s compared VAC, VAC plus prophylactic whole-lung irradiation (WLI), and VACA, and demonstrated the superiority of the VACA arm over the other arms [The first of these trials evaluated two combination therapies, controls . Subsequcontrols ,205,206.her arms   and the Pediatric Oncology Group demonstrated response rates ranging from 25% to 94% for ifosfamide or ifosfamide and etoposide (IE regimen) ,211,212.2, after which actinomycin-D was substituted. The five-year EFS for patients with non-metastatic disease in the experimental arm was 69% compared to 54% in the standard arm, establishing VDC/IE as the standard chemotherapy regimen in North America. In Europe, the EICESS-92 trial evaluated vincristine, dactinomycin, ifosfamide and doxorubicin (VAIA) neoadjuvant therapy with a randomization following local control in which standard risk patients were randomized to VACA vs. VAIA adjuvant therapy and high-risk patients were randomized to VAIA vs. VAIA plus etoposide [Subsequent cooperative group trials incorporated IE into VACA-based chemotherapy backbones ,217,218.toposide . There wtoposide   compared with conventional chemotherapy in localized EwS , both thPatients with isolated pulmonary metastatic disease were randomized to either six courses of VIDE and one cycle of VAI prior to either BuMel or seven courses of VAI and WLI. In this trial there was no clear benefit to BuMel over VAI and WLI . The EurThrough the EURO EWING Consortium (EEC), a harmonized protocol in Europe is targeted with iEuroEWING delivering a VDC/IE backbone , combineSuccessor trials have built upon these existing backbone regimens to add new chemotherapy combinations. Two North American trials have investigated the addition of topotecan and cyclophosphamide for newly diagnosed EwS. A phase 2 trial, COG 9457 evaluated an initial window of topotecan alone and in combination with cyclophosphamide . While tA number of trials have investigated the use of platinum-containing regimens in the treatment of patients with newly diagnosed EwS. The ISG/SSG IX protocol utilized alternating cycles of vincristine, doxorubicin, and ifosfamide, with cisplatin, doxorubicin, and ifosfamide, with a five-year EFS rate of 58%, inferior to existing contemporary regimens . The BraCare of patients with relapsed EwS remains a significant challenge and must be individualized for each patient’s disease and goals of care. Outcomes for this group of patients remain poor, with less than 25% being long-term survivors ,233,234.Most patients with first recurrent disease are treated with conventional systemic chemotherapy. Patients who demonstrate response to therapy may undergo local control to sites of recurrence, with either curative or palliative intent . Multiple chemotherapy regimens have been evaluated for efficacy in the setting of relapse and are now considered effective options at first relapse. For patients with either a particularly low chance of cure at first recurrence , or patients with second relapse and beyond, additional lines of therapy can be considered for palliation. These patients are also often candidates for phase I or phase II clinical trials of novel agents or combination therapies.Multiple systemic therapies have been evaluated for patients with relapsed disease, including regimens containing agents utilized as part of front-line therapy. Particularly for patients with late relapse, chemotherapy regimens may utilize a combination of agents that were included in front-line therapy as well as new agents with a reasonable chance of response ,237. ForTwo camptothecin-based regimens are used in clinical practice today: topotecan and cyclophosphamide; and irinotecan and temozolomide, with or without vincristine. Topotecan was not found to be as efficacious as monotherapy in this context ,241. How2/course) [The use of high-dose ifosfamide has demonstrated efficacy in patients with relapsed EwS, even following ifosfamide-containing front-line regimens. As noted previously, ifosfamide was identified to be an active agent against EwS in the 1980s ,211,212./course) . High-do/course) . An amenThe combination of gemcitabine and docetaxel has been evaluated in multiple trials, with response rates of 14% to lower doses of each agent , and 66%In patients who respond to conventional second-line chemotherapy, additional high-dose treatment may contribute to further reduce the risk of further events in patients . Both BuFew trials have evaluated maintenance strategies in EwS. The Italian Sarcoma Group (ISG)/AIEOP EWS2 Study evaluated the addition of maintenance oral cyclophosphamide and celecoxib for patients with metastatic EwS. This study included 71 patients who enrolled between 2009 and 2019, who received induction therapy, radiotherapy and/or surgery, followed by consolidative high-dose busulfan/melphalan + autologous stem cell rescue, whole-lung irradiation (12–15 Gy), and maintenance therapy for 180 days. The three-year EFS was 79% for lung or single bone metastatic disease and 19% for patients with multicentric metastatic disease. Follow up is ongoing.A maintenance strategy with zoledronic acid was evaluated in the Ewing 2008 trial. Patients with standard risk localized EwS did not benefit from maintenance treatment with zoledronic acid as add-on to the VIDE/VAI/VAC backbone. The three-year EFS was 84% in the zoledronic acid add-on group compared to 82% without zoledronic acid, while addition was associated with a higher rate of renal toxicity .Finally, the Latin American Pediatric Oncology Group conducted a trial studying vinblastine and oral cyclophosphamide as maintenance therapy in EwS, with results pending at this time.No studies thus far have demonstrated definitive benefit to maintenance therapy in EwS.The therapeutic success of chemotherapy in EwS has been remarkable if you compare today’s survival with that of the 1970s. However, no new drugs have been successfully introduced to newly diagnosed patients for almost 40 years. Fortunately, OS for patients with localized disease is approximately 80% based on optimization of the currently used chemotherapy drugs, such as interval compression. However, we have pushed our current regimen to the maximum tolerable intensity, while still failing to cure many patients and leaving survivors with a significant burden of late effects. Exploiting mechanism-based vulnerabilities appears the only way to further improve outcome or reduce the burden of dose-intense, side-effect prone chemotherapy.STAG2 (17%) or mutations/deletions of TP53 (10%). Despite this, a number of kinase inhibitors have been tested in EwS, but none of these have been successful in phase 2 evaluation, including inhibitors against aurora kinase A [A mechanism-based approach would utilize tumor-driving biologic insights that could include activating proto-oncogenes, inactivation of tumor suppressors or use tumor-specific genomic abnormalities to target toxin-bound antibodies or direct an immune-response to the cancer cell. The genomic landscape analyses of EwS demonstrate a paucity of recurrent mutations ,263. In kinase A , c-kit [kinase A , and insFew trials have evaluated targeted therapies for patients with newly diagnosed EwS. While no activating mutations are observed , the IGFAt this time, effective targeted therapies for patients with EwS are in development, but none are currently part of standard therapy. While sequencing of tumors should continue to be pursued, tumor sequencing at relapse typically does not yield targetable genomic lesions aside from the canonical fusions that heretofore have not been targetable ,263,271.Cabozantinib, a multi-tyrosine kinase inhibitor targeting primarily VEGFR2 and MET was recently evaluated in patients with relapsed EwS. MET signaling has been shown to be important in EwS tumorigenesis, and VEGF signaling for growth and metastatic potential ,273. A cMultiple studies have demonstrated that EwS tumors harbor deficiencies in DNA repair similar to BRCA 1/2 mutant cancers ,278. PolEWSR1 gene. EwS growth depends on the presence of the FET-ETS fusion [Clearly, the genomic marker that unifies EwS is a translocation that involves the S fusion ,282,283.S fusion ,285. EWSS fusion . These tDespite the attractive nature of targeting EWSR1-FLI1 fusion proteins, targeting the fusion is considered challenging as these proteins lack enzymatic activity and unlike kinases mostly lack obvious pockets for small molecules to bind . TherefoTK216 is a first in class small molecule inhibitor designed to directly inhibit the interaction between the ETS-family fusion partner  and RNA Helicase A (RHA), directly inhibiting the activity of the classical EwS fusion protein . TK216 iIndirect targeting of EWSR1-FLI1 has been hampered by the absence of a detailed understanding of the mechanisms of action of the fusion. That has impaired development of indirect targeting of drugs to inhibit EWSR1-FLI1 and its cellular pathways . Those cLaboratory-based work has also elucidated novel functions of trabectedin to inhibit the EWSR1-FLI1 transcriptional program in a schedule-dependent manner . A new cEffective clinical therapeutic targeting of EWSR1-FLI1 will require a deeper knowledge of its collaborating macromolecules . The mechanisms of how these macromolecules interact and modulate cellular programs that drive malignancy are at the forefront of research. Knowledge gained from continued cellular and animal models combined with training from clinical trial outcomes will be essential if patients with EwS are to have improved outcomes. The fact that targeting EWSR1-FLI1 is now in the clinic no longer relegates it to the ‘undruggable’ protein category.Many sarcoma patients in low- and middle-income countries are treated in hospitals lacking key infrastructure, including diagnostic capabilities, imaging modalities, treatment components, supportive care, and personnel. Abandonment and treatment-related mortality are additional challenges that complicate effective treatment and contribute to poorer outcomes of affected patients ,304. TheIt has been known for long that presence of clinically overt metastases at either diagnosis or at relapse constitute the strongest adverse prognostic factor in EwS, and survival of patients with primary metastatic or relapsed disease is similarly bad. Yet, primary and secondary metastatic disease are not quite the same. Primary therapy-naïve metastases arise in untreated patients from disseminated tumor cells before diagnosis. Experience from the pre-chemotherapy era taught us that resection of the primary tumor-mass alone does not prohibit disease progression, and almost every EwS patient will develop metastases during the course of disease in absence of any systemic therapy. Thus, it is assumed that micrometastatic disease is generally present at diagnosis, but chemotherapy is able to eradicate disseminated tumor cells in the approximately 70% of patients lacking evidence of metastases at diagnosis . This maEven though EwS is most frequently diagnosed at adolescent or young adult age, much later than typical pediatric cancers, the frequency of somatic mutations is among the lowest in all cancers with on average only 6 to 11 somatic mutations ,263,271.TP53 mutation, in bulk tumor analysis was found associated with inferior overall survival and time to progression [STAG2 loss was subclonal in the primary tumor, but characterized the main clone in the metastases. When viewed together with other studies, STAG2 mutation, present in about one fifth of EwS cases, may endow EwS cells with a more stem-like metastatic capacity [If the concept of early divergence and independent clonal evolution of metastases in metastatic EwS is correct, we might expect to see increased intra-tumor heterogeneity in the primary lesion of metastatic cases. This hypothesis received support from epigenome profiling of a large number of primary EwS. Here, intra-tumor DNA-methylation heterogeneity was higher in primary metastatic disease than in localized cases . Additiogression . In somecapacity ,313.Since only about 20% of patients with primary localized disease relapse with distant metastases, it has been speculated for long that there may be a difference in gene expression of primary tumors that tend to relapse as compared to those that can be successfully cured. Until recently, however, it has been difficult to perform systematic studies on metastatic and relapsed disease, since standard-of-care for EwS does not typically involve re-biopsy of the cancer when the disease returns or has metastasized (please see section “Biopsy—the holy tissue grail”). Therefore, most comparative investigations of primary tumors with and without progression, and metastases were based on patient cohorts too small to allow for meaningful conclusions and used Affymetrix oligonucleotide arrays to analyze differences in gene expression patterns at low stringency. In each of these investigations, similar numbers of up- and downregulated genes were described, but with little overlap between individual studies. Results of these studies are briefly summarized in The by far largest series of primary EwS analyzed for differential gene expression included patients from multi-centric American Children’s Oncology Group trials and European Intergroup Cooperative Ewing Sarcoma trials . SuperviIn addition to stromal infiltrates, the immune contexture of EwS may affect disease progression. Regarding density, type, and distribution of infiltrating immune cells, considerable inter-tumor variation was reported. Results of these studies are briefly summarized in Finally, gene expression profiling of peripheral blood cells in patients with EwS versus normal controls revealed that monocytosis and abnormal expression of CDH2 and CDT2 genes in the blood significantly correlated with poor patient prognosis, suggesting a systemic component in EwS disease progression, potentially through the intensification of osteoclastogenesis .In recent decades, therapies that stimulate host immune responses to tumors have begun to produce significant clinical results, for hematologic malignancies and, increasingly, for solid tumors as well. Such therapies include immune checkpoint inhibitors (ICIs) such as anti-PD1/PD-L1 or anti-CTLA4 antibodies and cell-based therapies such as adoptive T-cell transfer and chimeric antigen receptor (CAR)-T cells . InitialThe ganglioside GD2, well-established as an immunotherapy target in neuroblastoma, may also have promise in EwS, when combined with other agents such as all-trans retinoic acid, EZH2 inhibitor or antibody targeting HGF ,333,334.Altogether, so far bulk gene expression analyses did not reliably identify any EwS cell-intrinsic gene expression patterns discriminating between primary tumors of patients with localized disease, those presenting with metastases, and those at risk of treatment refractory metastatic relapse. However, these studies highlighted heterogeneity in the stromal and immune compartment affecting prognosis in EwS, and identified some candidate signaling pathways involved in potentially prognostic tumor microenvironment interactions.ICAM1, which was recently suggested as specifically associated with EwS invasion and metastasis [low cells are more chemoresistant, upregulate immune modulators PD-L1 and PD-L2, are resistant to T-cell mediated apoptosis, and upregulate angiogenic switch genes in response to Wnts [low cells [low tumor cells were estimated to exist in primary EwS at a frequency of about 1–2% [low cells in primary ES, but also elucidate potentially targetable interactions between distinct tumor compartments driving EWSR1-FLI1 fluctuations and EMT.Beyond inter-individual variation, the similarity of bulk gene expression patterns of primary tumors and corresponding metastases may not come as a surprise, as both disease stages are characterized by high proliferative activity. The major driver of tumor cell proliferation is the EWSR1-FLI1 oncogene present in most EwS cells of patients carrying the fusion. Experimental depletion of the fusion gene product results in loss of tumor cell proliferation but gain of migratory, invasive and metastatic properties as a result of epithelial-to-mesenchymal transition (EMT)-like cellular reprograming ,335,336.tastasis ,317. In  to Wnts ,338. We ow cells . In lineow cells . Using eout 1–2% . Recentlout 1–2% . So far,About one-quarter of EwS patients present with evidence of metastasis at diagnosis , and theWhat mechanisms explain the worse outcomes associated with metastasis at certain sites? In part this may be attributed to differential ability to apply therapy localized to the metastatic site. In the Euro-E.W.I.N.G. 99 study, patients receiving local treatment of both primary and metastatic sites had improved EFS compared to those who received either no local treatment, or local treatment of primary tumor only . SeveralBeyond these clinical features, however, there is the strong possibility that different features of the local tumor microenvironment in different anatomic sites may modulate the response of metastatic cells to treatment, including systemic therapies. These metastatic niches differ significantly in their biophysical properties and cellular composition, which may differentially impact on the clinical behavior of metastatic disease, as the niche environment is well known to influence the invasive and metastatic potential and response to treatment of a tumor. The non-cellular solid component of tissue provides structural integrity and supports biochemical signaling, while its mechanical properties regulate how the host tissue contains or restrains the tumor . The equlow EwS cells [Another biophysical factor discriminating between lung and bone niches is oxygen tension. While the lung is a highly oxygenated soft tissue which may elicit mutagenic oxidative stress in tumor cells homing to it , bone anwS cells  contribuwS cells . HoweverwS cells .S100 gene expression promoting lung colonization of tumor cells [On top of potentially chemokine-mediated tumor cell attraction by the metastatic niche, organotropism of metastases may be prepared by tumor-derived exosomes that carry a reprograming RNA and protein cargo to recipient cells at the site of metastasis. Lung specificity was reported to be mediated by specific α6β4 and α6β1 integrin patterns on the exosome surface, and exosome integrin uptake by lung resident cells resulted in activation of Src phosphorylation and pro-inflammatory or cells . Integrior cells ,364, butBone tropism of EwS metastasis is shared with common cancers such as of the breast, prostate, lung and kidney. Here, interaction with mesenchymal stromal cells in the metastatic niche may elicit a state of dormancy through BMPs and growth-arrest specific-6 (GAS6) protein secreted by the niche, permitting tumor cells to escape from anti-cancer drugs and immune therapy and emerge years to decades later as a clinically detectable bone metastasis ,366. In Upon homing to the bone/bone marrow, tumor cells disrupt physiological bone remodeling through the release of cytokines that stimulate osteoclastogenesis and resorption of the bone matrix and localized release of cytokines and growth factors , which further stimulate tumor cell proliferation . BisphosTaken together, the mechanisms supporting colonization of the lung and the bone by metastatic EwS may differ significantly with consequences for the treatment. It remains unclear, if there is a tumor-intrinsic (epi-)genetic component determining organotropism of EwS metastasis, which may serve diagnostic staging. Orthotopic xenotransplantation models are required to address a potentially patient-specific tumor-inherent preference of primary EwS to metastasize to either the lung or bone, as conventional subcutaneous implantation models usually fail to metastasize at all . AlternaTo improve outcomes for patients with metastatic EwS, preclinical models are critical, to support mechanistic studies of metastasis biology, and as a platform for testing new therapeutic approaches. Patient-derived cell lines have been a mainstay of EwS research, and a number of well-characterized cell lines are available and in wide use . While iThere is widespread interest in the development and use of patient-derived xenografts (PDXs) as an alternative to long-established cells lines. In addition to more accurately representing the genetics of the primary tumor (at least during early passages) , PDXs maA different approach is needed to truly recapitulate the complex physiology of tumor cell interactions with the non-tumor microenvironment. A number of systems have been employed to allow in vivo preclinical modeling of EwS metastasis. One study focused on the contribution of focal adhesion kinase to EwS cell migration via its effects on focal adhesion formation and Rho-dependent cell migration. These investigators employed an avian chorioallantoic membrane model for in vivo testing of the effect of FAK1 inhibitors on EwS cell growth . AnotherMouse xenografts have been widely used for in vivo studies of metastasis. The most straightforward approach is the introduction of tumor cells into immunocompromised mice via tail vein injection, allowing seeding of metastatic sites, most commonly the lung. Using this technique, it was demonstrated that Caveolin-1 contributed to metastasis via regulation of matrix metalloproteinase production and activation and expression of SPARC . SimilarAs a more natural model of metastasis, several studies have employed orthotopic xenografts. Tumor cell invasion and dissemination from a primary tumor established via intrafemoral injection was studied using multiple imaging modalities complemented by bioluminescence . Using aTo fully mimic the in vivo environmental factors determining metastatic potential, including the 3D environment, stromal tissue mechanical factors, the role of vascular and lymphatic compartments and the host immune system, a genetic animal model of EwS would be highly valuable as a complement to patient-derived in vitro models and xenografts. However, multiple attempts to create genetically-engineered mouse models of EwS have not yielded a tractable model, likely due to the developmental toxicity of heterotopic expression of the oncofusion . A zebra18F-FDG-PET/CT as a prognostic tool in EwS [18F-FDG-PET uptake shows a correlation between pre- and post-therapeutic standardized uptake value (SUV) and histological findings while providing data on additional quantitative parameters, such as metabolic tumor volume (MTV) and total lesion glycolysis (TLG) [Proteins and metabolic products in the blood, which support tumor diagnosis and allow early detection of relapse such as AFP in hepatoblastoma and germ cell tumors or catecholamine metabolites in neuroblastoma, are not yet identified in EwS. Instead, direct tumor components that are released into the blood as a liquid biopsy in EwS like circulating tumor cells (CTCs), circulating tumor DNA (ctDNA), circulating tumor RNA, and extracellular vesicles  containing tumor-specific biomaterials or signatures of the tumor microenvironment have been evaluated for their potential in clinical practice  402,403403. Progl in EwS . Responsis (TLG) ,405. StiThe prime and ongoing goal of biomarker research in EwS focus on identification and validation of biomarkers to stratify patients into specific clinical groups of EwS with similar prognostic outcome , or which can help modulate therapy approaches  towards a more personalized medicine approach. Especially in the young(er) patient collective, better selection of risk groups is essential to avoid treatment-related morbidity for survivors.EWSR1-ETS translocations—A clear target, with both direct and indirect strategies” and “EWSR1-FLI1 oncogene fluctuations as metastatic drivers in EwS”). Nevertheless, the complete and constant presence of the underlying chromosomal translocation currently appears to be the most advanced marker even if its use as an individual molecular biomarker appears to be very resource intensive. However, alternative ctDNA-based methods using low-coverage whole-genome sequencing will emerge in the coming years and could be used in the clinic as a minimal residual disease proxy  but these experiments have so far not converged into a common and consensus set of biomarkers . Anotherse proxy .Despite significant efforts in both diagnostic and therapeutic strategies, many aspects of EwS remain elusive, emphasizing on the ongoing need for translational, international-coordinated and joined research. Currently, clinical decision-making balances between protocol-driven regimen, clinical facts, and interindividual concepts.Recent decades have shown that tumor-specific strategies are difficult to implement. It is still non-directed chemotherapy controlling the disease for many patients but failing for far too many others. In the near future, a more personalized, translational approach embedded in prospective trial concepts is desirable and, even in the absence of targeted therapeutics, could mean personalize “treatment” without actually personalizing treatment.