Panthera spp.), a taxonomic group including tigers, lions, jaguars, leopards and snow leopards, is a daunting challenge. As expanding human populations across Panthera range countries exacerbate competition for land and prey, conflicts between humans and big cats are inevitable. Through a systematic review of the peer-reviewed literature published from 1991 to 2014 and indexed in Web of Science and Google Scholar (186 articles), our study explored the current state of knowledge regarding human-Panthera conflict and potential solutions, examining variables such as spatial and temporal distribution of research, methods used to study conflict, evaluation of interventions, and management recommendations. Our synthesis revealed several key data gaps and research needs. More studies could utilize diverse data collection approaches to focus on both the ecological and socio-cultural context for conflict. Additionally, only 21% of articles included in the review evaluated conflict mitigation interventions, and few of these yielded conclusive results. Success ratios suggest that compensation schemes and livestock management strategies were more effective tools for addressing conflict than either direct interventions  or community interventions . More studies should systematically evaluate the efficacy of conflict mitigation strategies, many of which are consistently recommended without empirical support. Results highlight trends and opportunities that can be used to inform future research and management efforts focused on human-Panthera conflict, ultimately enhancing the potential for coexistence between humans and carnivore species worldwide.Conservation of big cats ( Panthera spp.), a taxonomic group that includes tigers, lions, jaguars, leopards, and snow leopards, are apex carnivore species that drive the structure and function of biological communities in diverse ecosystems around the world [Panthera tigris) are classified as ‘endangered’ with a population of 3,200, lions (Panthera leo) are classified as ‘vulnerable’ with worldwide populations <30,000, jaguars (Panthera onca) are classified as ‘near threatened’ with worldwide populations of about 18,000, and leopards (Panthera pardus) are classified as ‘near threatened’ with worldwide populations unknown [Big cats  whose level of conflict with humans has been rated as high  or severe  [Panthera conflict research?; and (2) Which human-Panthera conflict mitigation strategies have proven to be most effective?Enhanced sharing of information across disciplines and geographies could help to resolve this complex problem. For example, although many studies have examined different aspects of the contentious relationship between humans and big cats, few have attempted to describe lessons learned from multiple social and ecological perspectives across space and time . Throughleopard) . We sougPanthera conflict in two comprehensive databases of scientific publications (Web of Science and Google Scholar) in February 2015. To be included in the review, a journal article’s title or key words had to contain at least one of the five Panthera species names (or common names) and at least one of the following words or phrases: attack, attitude, coexistence, human-wildlife conflict, or livestock  did not focus explicitly on at least one Panthera species, or (b) did not examine interactions between humans and the focal species. For example, studies with an exclusive ecological focus such as species ranges or prey selection and studies that did not assess or evaluate conflict with humans were removed from the analysis , the potential sample was reduced to 783 . A list of specific codes was compiled for topical categories until saturation was reached. Twenty interventions and recommendations that aimed to mitigate human-Panthera conflict were identified.To characterize human-Panthera species (most commonly involving leopards due to range overlap), the data from that article were included in results for both  species. In addition, some relevant studies of human-Panthera conflict that were not species-specific (i.e. literature reviews) were also included in the review.Using content analysis, two researchers then coded a subsample of 25 articles independently without knowledge of each other’s assigned codes following recommendations by Creswell  to increWe coded each of the articles for the following general categories ?Purpose of Study: How did the author(s) define the purpose of their study? The purpose of the article and type of conflict being studied were coded based on the purpose stated by the author(s) .Data Collection Methods: Were the data collected using social science methods , ecological methods  or a combination of these methods (coded as “hybrid”)?Type of Impact: What type of impact  was being studied? The type of impact was inferred by the researchers based on the results of each study.Evaluated Interventions: What conflict mitigation interventions, if any, were evaluated by the authors? Interventions were stated by the author(s) in the methods and/or results sections. Researchers categorized the interventions based on details provided by the author(s) , compensation schemes (proactive or reactive payments), community interventions  and direct intervention .Recommendations: What recommended conflict mitigation strategies were ultimately identified by the authors? Recommended interventions to reduce human-Panthera conflict were stated by the author(s), usually in the Discussion and/or Conclusion sections, and were based on either (a) the explicit evaluation results reported in the study (if applicable), (b) the expert opinion of the authors, or (c) some combination of the two.and recommended the same intervention strategy. We assumed that, based on the objective-centered approach frequently used to characterize program success in evaluation research [Because indicators of success varied across these interdisciplinary studies and effect sizes were rarely reported, a systematic quantitative comparison of intervention efficacy was not possible. We therefore assessed the efficacy of interventions by calculating subjective success ratios to determine the percentage of articles that both evaluated research  authors Panthera conflict identified by each study, but this proved to be challenging. For example, habitat loss and resource competition are closely linked to factors such as livelihood structures  and environmental policies and practices [We encountered several challenges with regards to data collection and coding for this review. We initially intended to assess the causes of human-ractices , making It should also be noted that all articles reviewed were treated as independent studies, even though a few study sites appear to have yielded multiple related articles from the same group of researchers. Additionally, it was difficult to account and control for both social and statistical heterogeneity within our analysis, which integrated studies using various forms of data collection in very diverse research contexts. Assessing the relative quality and/or validity of so many diverse studies was also challenging. By only focusing on published research, our study might also have failed to account for null results, generating a bias toward documentation of positive intervention effects. Because some degree of subjectivity is omnipresent in social science research, we elected to standardize analysis of studies based on how they were conceptualized and conveyed by the authors, not how they were perceived and interpreted by our research team. Overall, we feel that the selection and coding criteria described above allowed for objective analysis of the literature.Panthera conflict, these search terms may have inadvertently excluded tangentially related articles such as those focused on community-based natural resource management  or other conservation-oriented topics . Nevertheless, our search parameters generally paint a comprehensive portrait of the current state of research focused on human-Panthera conflict.Finally, three methodological limitations should be noted. First, our review only includes articles published prior to January 1, 2015. Since that time, the rapidly evolving body of literature on human-wildlife conflict has continued to progress, potentially yielding new insights not extensively reviewed here , 26. SecPanthera conflict highlighted study sites in thirty different countries (Our review of human-ountries . Distribountries . Sixty dPanthera conflict. The most common purpose (noted in 62 articles) was to simply assess the extent of conflict occurring. As human-Panthera conflicts vary in magnitude and severity around the world, it is not surprising that many researchers would aim to characterize the general nature of these interactions. A subset of these articles aimed to quantify the impact of conflict on either humans [The author(s) of the articles reviewed reported different reasons for studying human-r humans  or wild r humans , specifir humans  and evalr humans .Panthera conflict studies varied. Social science research strategies that centered on human responses such as interviews with key stakeholders (63 articles) and archives (67 articles)  appeared to be the most prevalent form of data collection. Although these social science methods were used for all species, the data collected did not always pertain to socio-cultural themes. For example, interviews and questionnaires were often used to obtain information related to species movements or livestock husbandry techniques, not psychological or cultural factors that might influence conflict. Ecological methods included direct observations of conflict incidents (40 articles) and a variety of tracking and monitoring tools. Radio collars were commonly used for lions whereas camera traps and field samples (e.g. scat) were more common for tigers and leopards. Studies using a combination of ecological and social science data collection methods were rare (29 articles) and were most common for studies focused on snow leopards.Data collection methods for human-Panthera range countries. Twenty-seven articles examined other impacts to human livelihood such as loss of property or income. These livelihood impacts were most commonly studied with regards to tigers (11 articles), snow leopards (8) and leopards (7). Loss of human life was most often studied with respect to tigers (15). More articles addressed injury or death to Panthera species than to humans. This was most common with regards to leopards (14 articles) followed by tigers (12) and lions (10). Only nine articles presented information related to the ecological impact of human-Panthera conflicts, and most of these focused on impacts to the prey base.The most commonly studied type of impact was livestock loss, which was addressed by 90 articles. These data are not surprising given the important role that livestock play in the livelihoods of people worldwide, particularly in Panthera conflict. Evaluations of interventions involving jaguars were particularly rare.Relatively few studies in the sample (n = 39) specifically evaluated conflict mitigation interventions. The most commonly evaluated interventions for almost all species fell into the category of livestock management strategies (34 articles), often focused on physical deterrents such as fences, dogs, and enclosed structures . ThirteePanthera conflict. The most commonly recommended interventions were improved livestock husbandry techniques  (54 articles), compensation schemes (44), and education (adult and/or youth outreach) (33). Livestock husbandry was recommended most frequently for lions and leopards whereas compensation schemes and education were recommended more in reference to tiger and snow leopard conflicts. Local management , and management/zoning  were recommended for all five species. Legal management  was presented as a recommendation more frequently for tigers than other species. Overall, recommendations encompassed a wide range of interventions–many more than were actually studied in our sample.Although only a small proportion of the articles we examined explicitly evaluated interventions, many of the articles issued specific recommendations for mitigating human-Because few studies systematically evaluated specific conflict mitigation interventions, it was difficult to draw definitive conclusions with regards to intervention efficacy. However, based on the evaluation studies that we reviewed, compensation programs and livestock management strategies  had the highest success ratios of 0.64  and 0.47 , respectively . SuccessPanthera conflict and the social forces  that influence it [Panthera conflict has increased substantially since the 1990s  were commonly used in the articles included in this study, but the information being gathered often focused on tangible metrics  and rarely accounted for underlying values, attitudes, and norms that may be driving behaviors [Panthera conflict [There are many ecological  and sociocultural dimensions  that contribute to the frequency and severity of conflicts in complex social-ecological systems , 53, reqehaviors . For exaehaviors  identifiehaviors . More reconflict , 26, 45.Panthera conflict. By understanding the movement, prey preferences, health, and ranges of animals, researchers may be better able to predict and ultimately prevent incidents of conflict. Ecological data collection methods used to study human-Panthera conflict include GIS/GPS [Panthera species included in this review, however, there is a significant lack of interdisciplinary research that integrates ecological and social science methods to paint a more complete picture of conflict and its effects on both humans and animals [A number of studies have also utilized ecological data collection methods to understand patterns of human- GIS/GPS , camera  GIS/GPS , field s GIS/GPS , and rad GIS/GPS . Some st GIS/GPS , 59–61.  animals . For exa animals  examine  animals , 65.Our review revealed a disconcerting finding with significant implications for big cat conservation practice: a noteworthy discrepancy between the number of conflict mitigation recommendations posited by researchers and the number of those interventions whose efficacy has actually been studied and/or systematically evaluated. Similar trends have been reported in other reviews of human-carnivore conflict , 26. IdePanthera conflict management.Overall, four categories of conflict mitigation strategies emerged through the review, demonstrating mixed results in terms of intervention efficacy. Given the small sample sizes and context-specific nature of intervention success , our calCompensation programs revealed the highest success ratios, and were most commonly studied with respect to snow leopards and tigers. Though they require financial resources that may not always be available, payment schemes that reward local people for conserving wildlife and wildlife habitat or, more commonly, compensate people for livestock loss with the hope of preventing the retaliatory killing of predators, can successfully help to secure coexistence between people and predators. In a review of financial instruments, Dickman et al.  found thPanthera cats as well. Our review suggests that compensation plans, though they might not be financially feasible in all contexts, have the potential to minimize retaliatory killings of predators while supporting local livelihoods.Another common challenge related to compensation plan implementation is that many are developed in relation to protected area boundaries. In reality, instances of conflict often occur outside these boundaries. Verifying conflict incidents and identifying who is responsible for compensating local people for wildlife damage outside protected areas (and across jurisdictions) is critical for the future success of this conflict mitigation strategy. The needs for enhanced communication within compensation programs to increase participation, improve response time, enhance transparency, derive fair compensation rates, and create opportunities for local management are commonly cited in the human-wildlife conflict literature , and seePanthera range countries due to extreme variation in climate and topography, knowledge of temporal shifts in depredation clearly aids the development of successful livestock husbandry techniques [Livestock management strategies were the second most successful types of intervention we studied. This category includes relocation of livestock or shifting herding patterns, fencing, dogs, water or noise deterrents, and other physical barriers. With limited funding and resources to devote to human-cat conflicts in locations around the world, refinement of livestock husbandry techniques may be among the most financially feasible and effective approaches to conflict mitigation, particularly when considering the prevalence of livestock predation among all big cat species . Evaluatchniques . UnderstSpatial management of livestock herds can also play an important role in limiting attacks and losses to carnivores. Herding near villages or areas of high human activity can limit incidents of conflict , 71 and Panthera cats. Hayward and Kerley [The use of dogs has been proven effective in limiting livestock losses to big cats with solitary lifestyles, including jaguars and leopards, as well as other cat species such as cheetah and puma in multiple contexts , 74. Desd Kerley  note thad Kerley .Panthera attacks on humans or other types of conflict.A focus on livestock management strategies is often the most beneficial, practical, and realistic mitigation method for communities that suffer from conflicts with predators . HoweverEfforts to address conflict by removing problem animals either by hunting, retaliatory killing, or relocation, appeared to achieve little success. Hunting was recommended most frequently for lions, possibly because of their unique appeal to conservation-oriented trophy hunters  or the hDirect interventions can also be carried out though translocation of problem animals. However, our review found limited instances where translocation was a success in mitigating conflict. For example, Athreya et al.  found thPanthera conflict or reviews of broader human-wildlife conflict [Our review revealed that documented success was also limited for community-based interventions designed to resolve conflict with big cats. These interventions include community-based natural resource management (CBNRM) programs, education and communication initiatives, a focus on local management and monitoring, ecotourism development, or legal management (implementation of policies or enforcement). Such initiatives can benefit communities in many ways , 81–83, conflict  either.Some research suggests that strategies designed to influence social interactions and cultural cognitions, including social marketing techniques  and educLocal management, which includes community involvement in decision making and strengthening of local leadership in response to conflict , was also highly recommended in our review, but rarely evaluated. Local institutional arrangements are key factors in setting up successful conservation and community programs, but variability in organization and institution structures must be taken into account , 81, 86.Panthera conflict and tolerance for predators.It should be noted that some successful elements of community interventions might have been inadvertently overlooked in our study due to the conflict-centered search terms. For example, specific components of social capital such as reciprocity, social networks, and stakeholder collaboration have been identified as critical to community actions to support or oppose tiger conservation outcomes in India  and MalaThis review suggests that, despite a rapid increase in research investigating human conflicts with big cats, many unanswered questions and opportunities remain. Some questions related to human-predator conflict have been addressed in studies published after our literature review was completed in December 2014 , 26, 34.Panthera conflict [Panthera conflict, particularly when compared to alternatives such as direct  and community-based interventions. Because so few studies have formally examined these strategies, however, it is difficult to draw definitive conclusions and identify best practices. Interventions must be both documented and systematically evaluated over the short and long-term to determine if they are or exacerbating or reducing conflict, ultimately impacting populations of humans and big cats. This is particularly true for community-based interventions such as education, community-based natural resource management, and legal management (policy enforcement and capacity), which are frequently recommended but rarely evaluated. As greater conflict mitigation emphasis is placed on promoting equity and sound governance in addition to technical fixes, investigations of community-based interventions will become even more important [Finally, our review echoes previous work and confirms that limited empirical evidence exists to inform recommendations for reducing human-wildlife conflict , 26–and conflict —across dmportant .Panthera conflicts, complementing research on human-wildlife conflict involving a broader array of taxa [Panthera conflict that may help multiple stakeholders achieve these goals.Our global assessment of research trends and opportunities reveals many insights that could be used to inform decisions, management plans, and future projects designed to address human- of taxa , 94. Loc of taxa . The nee of taxa , 97. ThiS1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.


In mammals, erythropoietin (EPO), produced in the kidney, is essential for bone marrow erythropoiesis, and hypoxia induction of EPO production provides for the important erythropoietic response to ischemic stress, such as during blood loss and at high altitude. Erythropoietin acts by binding to its cell surface receptor which is expressed at the highest level on erythroid progenitor cells to promote cell survival, proliferation, and differentiation in production of mature red blood cells. In addition to bone marrow erythropoiesis, EPO causes multi-tissue responses associated with erythropoietin receptor (EPOR) expression in non-erythroid cells such neural cells, endothelial cells, and skeletal muscle myoblasts. Animal and cell models of ischemic stress have been useful in elucidating the potential benefit of EPO affecting maintenance and repair of several non-hematopoietic organs including brain, heart and skeletal muscle. Metabolic and glucose homeostasis are affected by endogenous EPO and erythropoietin administration affect, in part via EPOR expression in white adipose tissue. In diet-induced obese mice, EPO is protective for white adipose tissue inflammation and gives rise to a gender specific response in weight control associated with white fat mass accumulation. Erythropoietin regulation of fat mass is masked in female mice due to estrogen production. EPOR is also expressed in bone marrow stromal cells (BMSC) and EPO administration in mice results in reduced bone independent of the increase in hematocrit. Concomitant reduction in bone marrow adipocytes and bone morphogenic protein suggests that high EPO inhibits adipogenesis and osteogenesis. These multi-tissue responses underscore the pleiotropic potential of the EPO response and may contribute to various physiological manifestations accompanying anemia or ischemic response and pharmacological uses of EPO. Erythropoietin is the hormone that regulates the daily production of 200 billion new red blood cells in the human body. Red blood cells are a renewable resource with a limited lifespan of about 120 days, have an intracellular protein content of about 95% hemoglobin, a tetrameric globular protein that binds oxygen cooperatively, and function primarily to transport oxygen from the lungs to the tissues. EPO binding to erythroid progenitor cells promotes their survival, proliferation, and differentiation to mature erythrocytes. EPO production is hypoxia inducible and is made in the interstitial cells in the adult kidney . In resp2-terminal 27 amino acid signal peptide and three potential N-linked glycosylation sites; posttranslational cleavage of the carboxyl terminal arginine gives rise to a 165 amino acid mature polypeptide for human EPO  stage that becomes the most responsive to changes in EPO level . EPO is e anemia . EPO bine anemia . The EPOe anemia . EPO bine anemia .Erythropoietin receptor is a member of the class I cytokine receptor superfamily that contains a WSXWS motif in the extracellular domain, a single transmembrane domain, a cytoplasmic domain that lacks tyrosine kinase activity and associates with JAK kinase, and form complexes that are homodimeric, heterodimeric or heterotrimeric . EPO binAvailability of recombinant human EPO facilitated discovery of EPO response in non-hematopoietic tissue. These include endothelial cells and the cardiovascular system, neural cells and the brain, myoblasts and skeletal muscle, adipocytes and fat depots, and bone ; .in utero, mice that lack EPO or EPOR exhibit angiogenic defects including decreased vessel networks  synthase (eNOS or NOS3) produces NO to regulate vascular tone and blood pressure. EPO stimulation of endothelial cells activates eNOS and NO production, particularly at reduced oxygen , 2011. TErythropoietin stimulation of endothelial cells increases ET-1 secretion that may exacerbate adverse vascular effects . Mice exDuring embryonic development, mice that lack EPO signaling exhibit ventricular hyperplasia indicating defect in proliferation and expansion of the myocardium . In studErythropoietin receptor expression in neurons and select locations in brain  combinedFor clinical application of EPO for ischemic injury, a Phase I clinical trial for EPO treatment of ischemic stroke provided evidence for the safety and potential efficacy showing an association with improvement in clinical outcome at 1 month . A folloHigh hematocrit resulting from chronically administered EPO, particularly at high dose, is associated with adverse effects such as hypertension and thromboembolism and could counteract the neuroprotective and cardioprotective effects of EPO. The potential to activate EPO/EPOR protective response in non-hematopoietic tissue via an alternate EPO receptor or an EPO mimetic without increasing erythropoietic activity and hematocrit is of particular interest. An alternate EPO receptor has been proposed for non-hematopoietic tissues such as brain and heart, consisting of a heterodimer between EPOR and the beta common (βc) receptor, also a member of the class I cytokine receptor superfamily and shared by receptors for granulocyte-macrophage colony stimulating factor and interleukins 3 and 5 . EPO proErythropoietin derivatives that can promote non-hematopoietic tissue protective effects, especially neuroprotection and cardioprotection, without stimulating erythropoiesis, have been proposed to bind alternate EPO receptors such as the EPOR/βc receptor heterodimer. These include asialoerythropoietin, carbamylated EPO, ARA 290 , and recombinant EV-3 (an EPO derived a spliced variant with exon 3 deleted) . Clinica+ satellite cells can self-renew or differentiate to Myf5+ committed muscle progenitor cells that contribute to growth, maintenance and repair of skeletal muscle  and type II (fast twitch) that differ in their function, mitochondrial density, and metabolic properties . Type I Erythropoietin regulation of metabolism extends beyond oxygen delivery and contributes to maintenance of white adipose tissue and metabolic homeostasis. Diet-induced obesity gives rise to glucose intolerance and insulin resistance, leading to type 2 diabetes. Animal studies suggest that EPO may be protective in diet-induced obesity, improves glucose tolerance, reduces insulin resistance and regulates fat mass accumulation, particularly in male mice .The anti-apoptotic and protective effects of EPO in select tissues such as adult and preterm brain contribute to an anti-inflammatory response, inhibiting expression of proinflammatory cytokines and reducing macrophage infiltration . The immE lack EPOR expression in white adipose tissue and exhibit higher circulating inflammatory monocytes on high fat diet feeding compared with wild type mice, suggesting a role for immune regulation by endogenous EPO/EPOR signaling   . These mE mice created on a C56BL/6 background exhibit no gross morphologic defects, they exhibit a disproportionate accumulation of fat mass with age , and in culture, EPO treatment decreases preadipocyte differentiation and induces ERK activation in primary mouse embryonic fibroblasts but not in embryonic fibroblasts generated from ΔEpoRE mice . Despitemulation .Insulin stimulates AKT activation and analysis of white adipose tissue from male C57BL/6 mice revealed that EPO treatment also increased AKT phosphorylation in white adipose tissue, but not in mice with targeted deletion of EPOR in adipocytes, suggesting that EPO modulates AKT activation in white adipose tissue and potentially affects insulin signaling . FurtherIn addition to EPO regulation of fat mass accumulation in white adipose tissue, EPO decreased lipid accumulation in the liver while stimulating STAT3/STAT5 activation and promoting lipolysis in white adipose tissue, suggesting benefit in non-alcoholic fatty liver disease . In browE mice show a decrease in STAT3 activation in hypothalamus and reduced POMC levels with and without EPO treatment, suggesting that EPO regulates appetite, and potentiates leptin response  and no association was found for EPO and percent weight change per year in the study group of 79 individuals (p = 0.02) (p = 0.02)  . In cont = 0.02) . Hence, In humans, as one ascends to high altitude increased EPO production via HIF regulation induces an EPO response that increases iron utilization for hemoglobin synthesis with expansion of red blood cell production with elevated blood hemoglobin and hematocrit . The relNote that high EPO levels can increase the risks of adverse effects such as cardiovascular complications and thromboembolic events, seizures, pulmonary embolism and death. For individuals in residence above 2500 m above sea level, about 5–10% can develop excessive erythrocytosis and associated Chronic Mountain Sickness . Howeverin vitro cultures  carrying BMSCs in immunodeficient mice model showed that BMSCs from ΔEpoRE mice that lack of endogenous EPO signaling results in reduced ectopic bone formation and increased marrow adipogenesis  to provide protection to ischemic stress or injury; skeletal muscle (myoblasts) for tissue maintenance or repair; white adipose tissue (adipocytes and macrophages) to protect from inflammation and from increase in fat mass in male mice during diet-induced obesity; and BMSC and osteoblasts to maintain normal bone development and bone remodeling accompanying exogenous EPO stimulated erythropoiesis . Sites oIn regulation of skeletal bone formation, endogenous EPO contributes to development and maintenance of skeletal bone and bone marrow adipocytes, and loss of non-hematopoietic EPO activity results in decreased bone formation and increased marrow adiposity . EPO admSex-specific differences in plasma concentration of EPO are not detected . HoweverTranslation of animal studies that demonstrate protective effects of EPO treatment in non-hematopoietic tissues to human disease manifestations can be problematic. Neuroprotection observed with EPO treatment in animal models of ischemic stroke and brain injury and the encouraging results in the Phase I clinical trial of EPO administration for ischemic stroke  did not All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fibromodulin (FMOD) is known as one of very important extracellular matrix small leucine-rich proteoglycans. This small leucine-rich proteoglycan has critical roles in the extracellular matrix organization and necessary for repairing of tissue in many organs. Given that the major task of FMOD is the modulation of collagen fibrillogenesis. However, recently observed that FMOD plays very important roles in the modulation of a variety of pivotal biological processes including angiogenesis, regulation of TGF-β activity, and differentiation of human fibroblasts into pluripotent cells, inflammatory mechanisms, apoptosis and metastatic related phenotypes. Besides these roles, FMOD has been considered as a new tumor-related antigen in some malignancies such as lymphoma, leukemia, and leiomyoma. Taken together, these findings proposed that FMOD could be introduced as diagnostic and therapeutic biomarkers in treatment of various cancers. Herein, for first time, we highlighted the various roles of FMOD in the cancerous conditions. Moreover, we summarized the diagnostic and therapeutic applications of FMOD in cancer therapy. Cancer is caused by uncontrolled division of abnormal cells which are able to invade other tissues. It is known as a result of an imbalance between cell proliferation and cell death rate caused by mutations in the regions of DNA which code regulator proteins . None isProteoglycans are relatively small components of ECM belonging to the small-leucine-rich-proteoglycan (SLRP) family , which aFMOD is a main proteoglycan which contributes to remodeling of the ECM through binding to matrix molecules, thereby plays an essential role in tissue repair, tumor progression and cancer . The intIt is believed that FMOD interaction with transforming growth factor (TGF)-β, a key profibrotic cytokine, keeps this growth factor within the ECM to regulate the action of local TGF-β Fig. . Due to In addition to its effects on the tumor cells behavior, TGF-β inhibits cell adhesion to the ECM, promotes extracellular matrix degradation, and induces immune suppression as well as angiogenesis. Through these mechanisms, TGF-β modulates the metastasis process . IncreasIn a study, Mayr et al., indicated that there were a significant up regulation of FMOD in chronic lymphocytic leukemia (CLL) cells than normal B lymphocytes . AuthorsGiven that FMOD and other members of the proteoglycan family could be involved in collagen fibrillogenesis and cell adhesion and they also help to suppression of tumor growth, regulation of cytokine activity, and prevention of apoptosis . FMOD haThe critical roles of FMOD in the pathogenesis of cancer had been ignored for a long time. Now, it is clear that the regulation of collagen fibrillogenesis and cell adhesion are not the only roles of FMOD and the other proteoglycans, but they also contribute to apoptosis prevention , 40. It NF-κB signaling is another pathway involved in apoptosis because its constitutive activation induces cell proliferation and inhibits apoptosis . Under nFMOD is an effective angiogenic factor with important roles in cell fate determination. FMOD plays a key role in angiogenesis in lung cancer, wound healing, optical and cutaneous diseases related to angiogenesis , 16, 43.These angiogenic regulatory effects strongly suggest that autocrine-secreted FMOD by cancer cells has a stimulating role in tumor angiogenesis of small-cell lung cancer. It facilitates the angiogenic phenotype transformation of endothelial cells as a pro-angiogenic step . The rol(GDNF) is a member of the TGF-β family. GDNF induces the expression of VEGF-C in neu-roblastoma cells and promotes angiogenesis in renal cells, normal skin cells, and hepatocellular carcinoma cells [NF-κB modulates the transcription of several target genes implicated in invasion angiogenesis, apoptosis, and migration , 47. It ma cells –52. Chenma cells  conducteTherefore, the main regulatory roles of FMOD in angiogenesis and apoptosis are associated with TGF-β superfamily as well as NF-κB signaling pathway. Consequently, new therapeutic medications could be produced to inhibit the development and progression of malignancies through regulating these pathways and direct interaction with mentioned agents.FMOD is regulated epigenetically through the methylation of its promoter. It induces the formation of filamentous actin stress fiber leading to promote the migration of glioma cells .The integrins have a main role in linking intracellular actin cytoskeleton to the extracellular matrix throughout cell migration process , 55. TheMondal and colleagues demonstrated that tumor secreted FMOD activates integrin-FAK-Src-Rho GTPase-dependent signaling to induce the migration of glioma cells. They also observed that the remodeling of actin cytoskeleton is critical for glioma cell migration induced by FMOD .Since effective treatments of early stages of diseases are available, new molecular biomarkers are urgently needed for an opportune diagnosis and adequate prediction of the clinical course of disease . The FMOFMOD has been recognized as a novel tumor-associated antigen in lymphoma, leiomyoma, and leukemia , 23. FMOIn a study, Choudhury et al., assessed the inhibition of FMOD and ROR1 in CLL cells . Given tIn recently study, Mondal and colleagues indicated that FMOD as a GBM over expressed gene because of the loss of promoter methylation . They shCurrent data suggest the important role of FMOD in the pathogenesis of cancer. This proteoglycan interacts with cellular and molecular mechanisms, such as NF-kB, to develop malignancy. FMOD acts through increasing the migration and angiogenesis to progress cancer. It also suppresses the apoptosis induction.Despite several attempts on this horizon, studies on FMOD are associated with some limitations. It seems that more pre-clinical experiments and clinical studies (with large samples) could contribute to more understanding of FMOD roles in the cancer pathogenesis. Moreover, more assessments could help to introduce this molecule as diagnostic or prognostic biomarkers for monitoring cancer subjects. This review suggest, evaluation of FMOD in all cancerous conditions may lead to introduce it to clinical settings.


Kaposi’s sarcoma-associated herpesvirus (KSHV) is the infectious etiologic agentassociated with Kaposi’s sarcoma (KS), primary effusion lymphoma, and multicentricCastleman disease. It has been shown that high KSHV prevalence and high incidence ofboth classic KS and AIDSassociated KS are found mostly among people of Uygurethnicity in Xinjiang, while people of Han ethnicity in Xinjiang have a higher KSHVseroprevalence than those of other Han populations in mainland China. However, it isstill unclear why there is such geographical and population variation in KSHVdistribution in China. In this work, we focused on the populations in the Kashgarregion and Urumqi area, where a total of 1294 research subjects were randomlyselected to investigate the potential correlation between KSHV prevalence anddifferent ethnicities in endemic areas of Xinjiang, and to determine risk factorsthat may affect KSHV infection rates or KS incidence. We identified a highseroprevalence of KSHV and high peripheral blood DNA infection in the general Uygurand Han populations in both Urumqi and Kashgar regions of Xinjiang, and determinedthat advancing age, low education level, and stationary population status affectKSHV infection rates. Further, KSHV-positive Uygur participants were shown to havehigher prevalence of neutralizing antibodies and neutralizing antibody titers thanKSHV-positive Han participants. Supplementary material is available for this article at 10.1007/s12250-017-4049-9 and is accessible for authorized users. Prevalence of Kaposi’s sarcoma-associatedherpesvirus in Uygur and Han populations from the Urumqi andKashgar regions of Xinjiang, China


This series on immunotherapies in acute myeloid leukemia (AML) aims to give readers new insights on established but also emerging immunotherapeutic approaches for AML patients. The therapeutic landscape in AML is rapidly changing, and several drugs have been developed and approved such as first and second generation FLT3 inhibitors ,2,3, IDHTherefore, immunotherapeutic approaches might be an option to prevent disease relapse and to eliminate leukemic cells or leukemic stem cells (LSC) that survive intensive treatment approaches. The efficacy of immunotherapeutic approaches has become ever more evident in solid tumors, especially immune-checkpoint inhibitors that are routinely used in several solid tumor entities, but also lymphoma ,12. Our focus in this special issue is different strategies of immunotherapeutic approaches in AML.Some of the immunotherapies in the treatment of AML, such as allogeneic hematopoietic stem cell transplantation (HSCT) and donor lymphocyte infusion (DLI), have been part of routine clinical practice in the treatment of AML for a long time, whereas other immunotherapeutic approaches have only recently entered clinical practice or need to be further developed. A key aspect is the mechanisms underlying the cure of AML patients, which are based on the graft-versus-leukemia (GvL) effect, in which allogeneic T cells recognize target antigens on malignant cells by T cell approaches including DLI. An effective and well-tolerated regimen for HSCT in patients with AML and MDS is the FLAMSA-RIC regimen, and therefore novel data of this approach are presented in this issue .It is very appropriate to utilize DLI after allogeneic HSCT to prevent relapse, to prolong progression-free survival, to establish full donor chimerism, and to restore the GvL effect in patients with hematological malignancies. There are different strategies to use DLI in a therapeutic setting for the treatment of morphological relapse, and also for prophylactic use in AML/MDS and DLI administered preemptively. There is also the approach of antigen-directed immunogenic and specifically stimulated and modified DLI as well as virus-specific donor T cells and third-party DLI .DC-based immunotherapies also have the potential to bring about demonstrable clinical responses in AML patients, although there has not been a complete breakthrough for this type of therapy until today. Van Acker et al. have highlighted different DC strategies in AML .Leukemia-associated antigens (LAAs) represent immunogenic structures to target LSC ,17, and In this special issue, we also elucidate antibody-based therapies in AML, such as T cell activating antibodies including immune-checkpoint inhibitors and diverse monoclonal antibodies ,12,24. IChimeric antigen receptor T cells (CARs) are highly effective in the treatment of refractory and relapsed acute lymphoblastic leukemia, to some lower extent in aggressive lymphoma, but also in multiple myeloma . HoweverAll of these aspects emphasize the high potential of immunotherapeutic approaches to improve the survival of AML patients in the future, where combination therapies utilizing immunotherapeutic drugs could represent further innovation strategies to further improve the treatment of AML.


Sex produces a new individual in which genetic material is reassorted and recombined. Most often, nuclear DNA is inherited from two parents, and organelle genomes are transmitted from only one of those parents. We report here genome-wide analysis of sexual recombination in social amoebae and show that inheritance in this protozoan is often triparental. Unusually, more than two gametes frequently fuse together and then split apart, allowing cytoplasm from multiple parents to be mixed. Progeny produced after two nuclei fuse and undergo meiosis thereby inherit nuclear DNA from two parents, and often mitochondrial DNA from a third. Our findings raise questions about mechanisms by which mitochondrial genes can promote self-interested organelle behavior during sex in social amoebae and other eukaryotes. Dictyostelium discoideum. We find that recombination occurs at high frequency in pairwise crosses between all three mating types, despite the absence of the Spo11 enzyme that is normally required to initiate crossover formation. Fusions of more than two gametes to form transient syncytia lead to frequent triparental inheritance, with haploid meiotic progeny bearing recombined nuclear haplotypes from two parents and the mitochondrial genome from a third. Cells that do not contribute genetically to the Dictyostelium zygote nucleus thereby have a stake in the next haploid generation. D. discoideum mitochondrial genomes are polymorphic, and our findings raise the possibility that some of this variation might be a result of sexual selection on genes that can promote the spread of individual organelle genomes during sex. This kind of self-interested mitochondrial behavior may have had important consequences during eukaryogenesis and the initial evolution of sex.Sex promotes the recombination and reassortment of genetic material and is prevalent across eukaryotes, although our knowledge of the molecular details of sexual inheritance is scant in several major lineages. In social amoebae, sex involves a promiscuous mixing of cytoplasm before zygotes consume the majority of cells, but for technical reasons, sexual progeny have been difficult to obtain and study. We report here genome-wide characterization of meiotic progeny in  Inheritance of nuclear genes largely follows the Mendelian laws of segregation and independent assortment. Individual eukaryotes are produced either clonally, having a single parent, or sexually, with two parents. In contrast, cytoplasmic genes display non-Mendelian patterns of inheritance and are typically transmitted only from one of the two parents during sex. The uncoupled inheritance of these different sets of genes creates recurrent genetic conflict –3. The eDictyostelium –13Dictyocan form , 15. Dicn occurs B and gron occurs C. Meiosin occurs , 16. Maces known , 18. In es known  has beenes known . How reces known , 22.D. discoideum mating types  and sequenced the genomes of haploid progeny clones along with those of their parents. The parental genomes were extensively polymorphic, so that after filtering of putative variants, thousands of sites could be used to assess recombination in meiotic progeny across the 34-Mb genome in each cross. The type II parent strain, HM597, is a cycloheximide-resistant mutant; initial observations confirmed that cycA, which lies on chromosome 1 . We found that progeny from each macrocyst are identical, implying that only one meiotic product survives in each cyst, as has been reported previously in some Dictyostelium species . However, different macrocysts in a given cross give rise to diverse progeny genotypes .To investigate these inheritance patterns in greater detail, we germinated macrocysts from three pairwise crosses between strains of all three mosome 1 , and the species , 25 . In this three-way cross, any lateral genetic contribution from the matA mutant cells into progeny can be detected by tracking sequence variants present solely in the mutant background.We reasoned that the unusual Spo11-independent recombination during sex in social amoebae could be connected with the conflictual nature of macrocyst formation, during which large numbers of “victim” amoebae are consumed ; more br progeny  . Across all progeny characterized in two- and three-way crosses, crossovers occurred on average approximately once per megabase in each meiosis , comparable in frequency with budding and fission yeasts .omosomes B. The siomosomes C. Where SI Appendix, Fig. S4). In Dictyostelium, the rDNA is maintained at high copy number as extrachromosomal palindromic molecules, and a chromosomal copy has been proposed to exist on chromosome 4  carries a high-copy nuclear plasmid that is stably carried by haploid cells during proliferation and asexual development .Ribosomal DNA, which is maintained in growing cells as multiple copies of a linear extrachromosomal element, was inherited largely uniparentally . Remarkably, however, in three of four sequenced progeny from three-way crosses, we found that mitochondrial genomes were inherited from the matA null cells . Using fluorescent proteins targeted to mitochondria and nuclei, we could visualize thorough mixing of cytoplasms in syncytia formed during three-way crosses, indicating that lateral transmission of mitochondria likely occurs via these transient fusions early in the sexual cycle . In the two-way HM597 × HM598 cross, 10 of 11 progeny carried predominantly the HM597 mitotype, and the other carried both mitotypes in near-equal quantities. For the three-way cross, the HM597 mitotype was predominant in 31 of 41 progeny, HM598 in five, and the matA null mitotype in two, whereas in three progeny, no single mitotype was predominant. In all five progeny in which the HM598 mitotype predominated, the matA null mitotype was present in small quantities . This indicates that mitochondrial inheritance is not strictly controlled by the mating-type locus in this species; similarly, biparental inheritance often occurs in myxogastrids, one of the sister groups of social amoebae . This suggests, consistent with earlier reports of transient anastomoses and transfer of cytoplasm between aggregating cells , as well as 20 other D. discoideum isolates for which whole-genome data are available . Two genes that are highly polymorphic relative to their size, atp4 and atp8, encode components of the stator stalk of ATP synthase , with long N-terminal extensions. Unusually, these genes have more nonsynonymous than synonymous changes , possibly as a result of positive selection. The D. discoideum uS3m protein is also striking because it appears to be split across two polypeptides, Mp26 and Mp27 (mp05, is specifically enriched in gametes (Our findings suggest strong sexual selection on mitochondrial genotypes might occur in social amoebae. As an initial approach toward assessing the genetic effects of this proposed selection, we cataloged the mitochondrial genome variants of the four parental strains of this study (the three wild-type parental strains plus the vailable . The mitsynthase . The twond uS11m  (SI Appeand Mp27 . We suggSex originated early in eukaryotic evolution before the last eukaryotic common ancestor, but most likely after the first mitochondriate common ancestor . Before We did not find evidence that the reconfiguration of meiotic recombination in social amoebae is connected to the cannibalistic transfer of resources during macrocyst formation. Our data suggest that crossover designation is nonrandom, but the unconventional mechanism that operates in social amoebae remains mysterious; we have no strong candidate inducer of DNA breaks and cannot rule out the possibility that spontaneous lesions are used to initiate recombination . We suggDictyostelium sex might affect the conflicts engendered by the dramatically different fates of cells during macrocyst formation. We predict further that important cell-biological consequences might follow from the mixing of many cells’ cytoplasms during sex, as it risks the spread of harmful endosymbionts, such as selfish mitochondrial genomes, viruses, and the bacteria present in certain isolates (Our findings on mitochondrial inheritance raise questions about how the strikingly non-Mendelian character of isolates , 42. To isolates .Dictyostelium amoebae were cultivated on bacteria, using standard techniques. The strains used in this study are described in SI Appendix, Table S1. Macrocysts were prepared by growth of mixed strains on LP agar plates submerged under buffer in the dark. The procedure for germination of macrocysts followed that of Wallace and Raper (SI Appendix, SI Materials and Methods. Care was taken to avoid the possibility of parental amoebae and spores being carried through to the germination plates; these can presumably become trapped among the fibrous material associated with macrocysts and, if bacteria from the growth plates are also present, can grow to greater numbers than the macrocyst progeny. Germination plates with unusually large numbers of fruiting bodies were invariably dominated by parental genotypes and were discarded. Details of methods for genome sequencing, read alignment and variant calling, identification of crossover sites, enumeration of mitochondrial and ribosomal DNA variants, PCR screening of mitotypes, generation of fluorescent protein reporter strains, and flow cytometry and fluorescence-activated cell sorting can also be found in the SI Appendix, SI Materials and Methods.nd Raper . A full Whole-genome sequencing data and consensus parental genomes are available under the accession PRJEB28008 .Supplementary File


Cholesterol is an essential structural component of cellular membranes. In addition to the structural role, it also serves as a precursor to a variety of steroid hormones and has diverse functions in intracellular signal transduction. As one of its functions in cell signaling, recent evidence suggests that cholesterol plays a key role in regulating angiogenesis. This review discusses the role of cholesterol in angiogenesis, with a particular emphasis on cholesterol trafficking in endothelial cell signaling. Small molecule inhibitors of cholesterol trafficking and their preclinical and clinical development targeting angiogenesis and cancer are also discussed. Cholesterol is an indispensable constituent of cellular membranes and plays a critical role in membrane permeability and fluidity. In addition to structural support, it also functions in intracellular transport and cell signaling as a critical component of lipid rafts ,2. UnderCholesterol homeostasis is tightly modulated by a complex network which involves its synthesis, import, export, esterification, and metabolism . In the Extracellular cholesterol (LDL) absorption and distribution into cells requires an appropriate endosomal trafficking system . LDL binExtracellular cholesterol (circulating LDL) in the blood vessels needs to enter or pass through the endothelium layer to be distributed to the whole body. Therefore, blood vessels and endothelial/vascular smooth muscle cells play an important role in whole-body LDL penetration, accumulation, and metabolism . Convershttps://clinicaltrials.gov). In addition to the cholesterol biosynthesis inhibitors, the body cholesterol absorption inhibitor ezetimibe has also been reported to inhibit angiogenesis. Ezetimibe (Zetia) is an FDA-approved, cholesterol uptake-blocking drug which works by binding to and inhibiting NPC1-like 1 (NPC1L1) protein, a gastrointestinal tract transporter responsible for dietary and biliary cholesterol absorption [In the late 1990s, several groups reported possible connections between cellular cholesterol levels and angiogenesis based on the observation that HMGCR inhibitors (statins) that inhibit cholesterol synthesis could modulate angiogenesis. Negre-Aminou et al.  reportedsorption . Solomonsorption  reportedCholesterol is an important component of lipid rafts in the cell membrane where the membrane proteins are anchored. Lipid rafts in the cell membrane function in cell signaling by serving as scaffold structures for signaling molecules . The depWe discussed how endothelial cell cholesterol levels affect angiogenic signaling. Depletion of intracellular cholesterol in turn reduces the amount of plasma membrane cholesterol that affects lipid raft structure and interferes with the major angiogenic receptor signaling on the cell membrane. In addition to the total cholesterol level, intracellular cholesterol distribution governed by cholesterol trafficking is also essential for proper functioning of lipid rafts in the plasma membrane and other cellular membranous organelles. We, and other groups, have identified several small molecule inhibitors of endothelial cell cholesterol trafficking and proposed a potential role of cholesterol trafficking in angiogenesis based on their pharmacological effects.Itraconazole is an azole antifungal drug approved for patients with fungal infections. From a screening for anti-angiogenic agents using a clinical drug library, Chong et al.  identifiSelective estrogen receptor modulators (SERM), such as tamoxifen, are estrogen receptor antagonists that have long been used for the treatment of patients with estrogen receptor-positive breast cancer since the 1980s. However, several studies revealed that tamoxifen also showed an anticancer effect in estrogen receptor-negative breast cancer ,44,45. LCepharanthine is an approved drug in Japan used for various illnesses such as inflammation and the alleviation of chemotherapy-induced adverse effects . In clinIn this review, we overviewed cholesterol synthesis, regulation, and distribution, and discussed the potential role of cholesterol levels and trafficking in angiogenesis based on experimental evidence. There are several cholesterol sensing mechanisms in cells, such as SCAP-INSIG-1-SREBP and squalene monooxygenase, to maintain proper cholesterol levels ,10,11,58In addition to VEGFR2 and mTOR, there are a number of membrane proteins that participate in angiogenic signaling. It cannot be ruled out that cholesterol depletion may also influence other signaling molecules on the membrane in endothelial cells. Further studies are required to clarify whether certain specific proteins, or all of the membrane proteins generally, are modulated in their functions upon cholesterol depletion.In summary, we, and other groups, have proposed that cholesterol trafficking inhibitors are potential anti-angiogenic agents based on their strong pharmacological activities. The cholesterol trafficking inhibitors introduced in this review are FDA (or foreign counterparts)-approved drugs that are well-tolerated in humans. Therefore, a repurposing of the drugs for angiogenesis or cancer treatment is actively ongoing. This study also supports the idea that endothelial cell cholesterol trafficking is a viable drug target for angiogenesis, thus providing a new target for screening and future drug discovery, and development for cancer treatment.


Genetic and pathophysiologic criteria prearrange the uncontrolled growth of neoplastic cells that in turn initiates new vessel formation, which is prerequisite for further tumor growth and progression. This first endothelial lining is patchy, disordered in structure and thus, angiogenic tumor vessels were proven to be functionally inferior. As a result, tumors were characterized by areas with an apparent oversupply in addition to areas with an undersupply of vessels, which complicates an efficient administration of intravenous drugs in cancer therapy and might even lower the response e.g. of radiotherapy (RT) because of the inefficient oxygen supply. In addition to the vascular dysfunction, tumor blood vessels contribute to the tumor escape from immunity by the lack of response to inflammatory activation  and by repression of leukocyte adhesion molecule expression. However, tumor vessels can remodel by the association with and integration of pericytes and smooth muscle cells which stabilize these immature vessels resulting in normalization of the vascular structures. This normalization of the tumor vascular bed could improve the efficiency of previously established therapeutic approaches, such as chemo- or radiotherapy by a more homogenous drug and oxygen distribution, and/or by overcoming endothelial anergy. This review highlights the current investigations that take advantage of a proper vascular function for improving cancer therapy with a special focus on the endothelial-immune system interplay. QuiPhenotypic differences at the molecular and functional levels have been identified for tumor and normal endothelial cells . Tumor sICAM-1 gene by reversal of histone modifications in the ICAM-1 promoter, thereby restoring leukocyte-vessel wall interactions and leukocyte infiltration  in tumor endothelial cells, which then causes the unresponsiveness of tumor endothelial cells to inflammatory signals, a phenomenon called endothelial cell anergy that causes lymphocyte tolerance –48. Hencltration .A functional vascular network is prerequisite not only for nutrients or oxygen supply but also for the immune cells to enter the tissues. The functional and structural abnormalities of tumor blood vessels together with the unresponsiveness of the endothelium to inflammatory stimuli caused by proangiogenic factors decrease the recruitment of immune effector cells into the tumor, thus limiting the effectiveness of cancer immunotherapies , 55. GivToday, immunotherapy for activating therapeutic anti-tumor immunity has become a mainstay of cancer therapy , 58. AltThe different approaches being currently explored to increase recruitment of immune effector cells, include manipulating the expression of homing-associated molecules on T-cells and tumor endothelial cells. Concerning the first option, a successful approach to target or restore tumor-induced immunosuppression was made by adoptive cell therapy using tumor-reactive T-lymphocytes that resulted in objective tumor regression in >50% of treated patients . The potThus, strategies addressing directly the vascular system to sensitize tumors or improve the therapeutic response in cancer therapy were established and already shown to exert beneficial effects in immune checkpoint blockade. In a very elegant preclinical study Elia et al. showed that a selective (pre)activation of the tumor endothelium with the cytokine TNF promoted intratumoral T-cell infiltration, and immune checkpoint blockade . The autProper vascular function as revealed by measurements of vessel perfusion was further used to predict the therapeutic response to immune checkpoint blockade . Here, tConclusively, tumor endothelial cells are actively involved in immune cell exclusion and inhibition of lymphocyte activation, fostering an immunosuppressive intratumoral microenvironment that contributes to the tumor immune escape and severely impairs conventional cancer therapies , 14, 69.Using regulator of G protein signaling 5-deficient mice, a genetically induced vascular normalization mouse model, in which newly formed blood vessels were characterized by a mature and thus stabilized phenotype, it was further shown that tumor vessel normalization consequently reduced vascular leakiness and hypoxia within the tumors, leading to an influx of immune effector T-cells , 30. HerInstead then of starving tumors from their blood supply and achieving complete vessel regression, vessel normalization by anti-angiogenic therapy has gained more attention for generating more mature and regular functioning tumor blood vessels with increased vessel perfusion. This is supposed to improve distribution of circulating blood components, oxygenation, removal of suppressive metabolites, as well as distribution of therapeutically applied drugs . In addiImproving the aberrant structural abnormalities and associated dysfunctionalities of tumor blood vessels, and thus lowering tumor hypoxia and enabling immune cell infiltration, by antiangiogenic therapy was shown to synergize with immunotherapies for more durable effects . In an pIn addition, to tumor endothelial cell anergy that limits the adhesion and subsequent extravasation of recruited leukocytes, tumor-derived factors can further induce endothelial cell-mediated apoptosis of recruited immune cells, e.g., by induced death mediator Fas ligand  expression which directly kills anti-tumor T-cells finally leading to an inefficient recruitment of effector CD8(+) T-cells into the tumor , 75. WitAnother molecule which became of interest for activating therapeutic anti-tumor immunity is the interferon-inducible intracellular enzyme indoleamine 2,3-dioxygenase 1 (IDO-1), which catalyzes the initial and rate-limiting step in the degradation pathway of the essential amino acid tryptophan to kynurenine , 81. KynHowever, the actively participation of tumor endothelial cells in the innate and adaptive immune responses is not limited to the ability to attract and direct a wide range of immune cells and elevate extravasation from the host circulation. Tumor endothelial cells are believed to have a role in antigen presentation , 13, 14.Conclusively these findings strongly argue for new therapeutic approaches including combinations of the anti-angiogenic treatments with immunotherapies in addition to the current standard regimens for cancers, particularly for those that do not respond to surgery, chemotherapy, or radiation.Tumor eradication or local cancer control for a better outcome are the main goals of radiation therapy. Endothelial cells act as critical determinants of the radiation response in tumors as radiotherapy generally fosters endothelial apoptosis, increased vascular permeability, and acquisition of a pro-inflammatory and -coagulant phenotype –95. The As an apparent approach, sensitizing tumor endothelial cells to radiation-induced apoptosis resulted in a more pronounced tumor growth delay upon irradiations in preclinical animal models, which suggested that a therapeutic targeting at the level of the tumor vasculature could counteract radiation resistance , 100. InTo improve blood perfusion and thus tumor oxygenation, again vascular normalization using anti-angiogenic-therapy was suggested , 104. AcIn general, the pharmacological inhibition of pro-angiogenic factors triggers apoptosis of angiogenic endothelial cells in the immature and leaky tumor blood vessels leading to the selection for mature, non-leaky vessels, the so-called pruning effect. Within these matured and normal vessels endothelial anergy is restored. Jaillet et al. reported that ionizing radiation altered the glycosylation pattern of endothelial cells, in particular increased high mannose-type N-glycans and decreased glycosaminoglycans, which stimulated the interactions between irradiated endothelial cells and monocytes . Thus, tIn addition, preclinical and clinical evidence exists for the immuno-stimulatory properties of radiotherapy. Radiation treatment can foster immunogenic tumor cell death whereby danger-associated molecular patterns  were released which in turn can recruit and activate dendritic cells to process tumor antigens for naïve T-cells finally resulting in an anti-tumor immune responses , 112. OfThe tumor vascular endothelium is a key cell compartment for the response of tumors to cancer therapy. The tumor initiated neovascularization for nutrients and oxygen supply prior tumor progression results in a structural and functional abnormal tumor vasculature, which contributes to a pro-tumorigenic and immunosuppressive environment altering the therapy response of tumor cells. In particular for clinically approved immunotherapies, such as immune checkpoint blockade and adoptive T-cell transfer, the functional abnormal tumor vasculature fosters therapy resistance by limiting an inefficient recruitment, distribution and infiltration of tumor eradicating immune cells. Therefore, tumor vasculature targeting agents in order to re-activate specifically the tumor endothelial cells in terms of vascular normalization provide promising strategies to optimize the efficacy of currently employed cancer therapies, especially immunotherapies.The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Receptor of urokinase-type plasminogen activator (uPAR) is the one of key players in cancer initiation and progression. uPAR interaction with its specific ligand urokinase (uPA) promotes cancer cell migration, invasion, and metastasis; whereas its lateral interaction with integrins and other transmembrane receptors, including EGFR and IGFR1, leads to initiation of intracellular signaling responsible for the activation of dormant cancer cells, epithelial-mesenchymal transition as well as enhanced survival and resistance to apoptosis of tumor cells. Pro-survival signaling activated by uPAR might be responsible for the escape of cancer cells from the cytotoxic effects of anticancer drugs  and tumoThese multifaceted roles of uPAR in cancers make it an attractive target for anti-cancer therapy. Various approaches to inhibit uPAR expression or activity, including small molecules, antibodies and siRNAs demonstated significant down-regulation of tumor growth due to reduced tumor vascularization, suppressed cancer cell survival and proliferation. Although suppression of uPAR by siRNA is effective, it is not stable and therefore may not be sufficient due to the rapid dilution of siRNAs in fast proliferating cancer cells .Plaur) in Neuro 2A neuroblastoma cells. Down-regulation of uPAR expression caused a reduction in proliferation of neuroblastoma cells accompained by their enhanced apoptosis. These data indicate that CRISPR/Cas9n technology could be feasible for uPAR silencing in neuroblastoma [Recent study by Rysenkova et al. scheduled for publishing in Oncotarget provides novel insights into the role of uPAR in neuroblastoma. Earlier, elevated expression of uPAR was found in human neuroblastoma and correlated with high invasiveness of this tumor . In the blastoma .Importantly, authors demonstrate that uPAR silencing correlated with a decrease in full-length TrkC mRNA expression, which could not be accounted for off-target effect of applied method. TrkC belongs to the family of tropomyosin-related kinase receptors, comprised of TrkA, TrkB and TrkC itself. These are transmembrane receptors with tyrosine kinase activity, which specifically bind neurotrophic growth factors. TrkC interaction with its ligand neurotrophin-3 (NT-3) provides pro-survival and proliferating stimuli for neural cells. However in the absence of NT-3 vacant TrkC induces apoptosis, behaving as a classical «dependence receptor». Such apoptosis activation in the absence or dramatic drop down of the ligand is important mechanism of limiting cell numbers during development and could be beneficial in tumorogenesis. Indeed, high expression of TrkC is associated with favorable outcome for neuroblasoma patients . Pro-surThese data also indicate that urokinase system is likely to be involved in the control of neural cell sensitivity to NT-3. Previously uPAR was shown to be involved in the control of neuronal cell migration and directed axonal growth . Data ob


Nanoparticles have seen considerable popularity as effective tools for drug delivery. However, non-specific targeting continues to remain a challenge. Recently, biomimetic nanoparticles have emerged as an innovative solution that exploits biologically-derived components to improve therapeutic potential. Specifically, cell membrane proteins extracted from various cells  have shown considerable promise in bestowing nanoparticles with increased circulation and targeting efficacy. Traditional nanoparticles can be detected and removed by the immune system which significantly hinders their clinical success. Biomimicry has been proposed as a promising approach to overcome these limitations. In this review, we highlight the current trends in biomimetic nanoparticles and describe how they are being used to increase their chemotherapeutic effect in cancer treatment. Cancer is the second leading cause of death in the world with over 8 million deaths worldwide. Current treatment option range from surgery and radiation to chemo and hormone therapy . AlthougInitially, our group engineered multi-stage porous silicon NPs to mimic red blood cells and serve as a carrier to shuttle loaded nanoparticulates . StrategIn an effort to mitigate immunorecognition and sequestration by filtering organs, our group modified multi-stage NPs with a leukocyte-derived cell membrane shell as a strategy to bypass critical biological barriers . Other gThe aim of this review is to highlight the various strategies in which biomimetic NPs are being used in cancer treatment. In addition, this review will cover the various cell sources employed for NP design and the intrinsic effects these cells provide in tumor targeting.Due to unique biological properties such as prolonged blood circulation time, lack of organelles , and abundance in the body, red blood cells (RBCs) represent the most convenient cell membrane protein source to produce cell-based NPs. In addition, thanks to the expression of specific inhibitory proteins such as CD47, also known as the “do not eat me” signal, RBCs can easily escape immune system recognition, inhibiting macrophage-mediated phagocytosis . Zhang aSimilarly, Su et al. formulated paclitaxel-loaded NPs using a polymeric core and a hydrophilic RBC vesicle shell  that were co-administrated with the tumor-penetrating peptide, iRGD, to enhance antitumor therapy . The autRecently, platelets have also garnered significant attention as a source for biomimetic NPs. Derived from the bone marrow, these enucleated cells are involved in hemostasis, clotting, inflammation, as well as tissue repair . SeveralTaking advantage of the interactions between platelets and tumor cells, and thanks to their physical and biochemical properties such as discoidal shape and flexibility, biomimetic platelet-like NPs have been exploited for targeted drug delivery . Li et aA) functionalized with an RGD peptide to specifically target integrins  within tumor blood vessels. The second was a biomimetic nanocarrier (NCB) made of integrated platelet membranes and loaded with paclitaxel (PTX). The authors showed that most of the proteins expressed on the platelet membranes, such as CD36, CD42d, P-Selectin, and CD40L were successfully transferred onto the NP surface. This transfer mediated NP accumulation at the tumor site, reduced blood vessel inflammation and increased drug accumulation and treatment efficacy in the tumor cells [A similar approach was used by Hu et al. that developed platelet membrane (PM)—coated core–shell nanovesicles  loaded with two anticancer components: TRAIL and DOX. The administration of PM-NVs in a breast cancer mouse model demonstrated NP accumulation at the tumor site and efficient delivery of TRAIL toward cancer cell membrane, resulting in the activation of the extrinsic apoptosis signaling pathway. Moreover, thanks to their acid-responsive encapsulation matrix, the PM-NVs were better digested after endocytosis, thus enhancing DOX intracellular accumulation. This resulted in the inhibition of tumor growth and a reduction in lung metastasis . The samor cells . Thanks or cells .It has been largely accepted that most current anti-tumor therapies fail as a result of a small cell population  responsible for tumor progression, tumor maintenance and metastases formation. CSCs have been identified and described in almost all types of cancer  are multipotent stem cells endowed with the ability to differentiate into osteoblasts, adipocytes, chondroblasts, fibroblasts and pericytes. Stimulated by microenvironmental factors such as hypoxia, ligands of Toll-like receptors or cytokine gradients within the extracellular matrix, MSCs migrate to injured and inflamed sites. With cancer being considered as a disease of chronic inflammation, it’s appropriate that the last decade has seen a considerable rise in MSCs used as a therapeutic tool in tumor treatment , 49.In a biomimetic strategy employing MSCs, Timaner et al. developed nanoghosts (NGs) derived from the cytoplasmic membranes of MSCs. MSC-NGs retained the ability of MSCs to target inflamed endothelium, allowing safe and effective targeted delivery of their payload both in vitro and in vivo using a prostatic mouse cancer model. In addition, the authors demonstrated that MSC-NGs accumulated in proximity of CSCs within the tumor mass. The membrane proteins expressed on the NG surface mediated cell-to-cell interaction with CSCs , thus suA similar approach was used by Machluf and coworkers that resulted in a significantly higher accumulation of MSC-derived NGs within the tumor as compared to smooth muscle cell-derived NGs . MSC-NGs that retained on their surface specific MSC integrins were shown to bind and fuse to the tumor cell surface and disrupt the plasma membrane, leading to a cytotoxic effect and tumor growth inhibition. The authors suggested that the anti-tumor effect observed using empty MSC-NGs was likely due to the interaction of NG with the different components of the tumor microenvironment . ConversOn the other hand, Gao et al. demonstrated that MSC-biomimetic NPs were endowed MSC-derived hypoimmunogenicity due to different molecular recognition moieties. Indeed, bone marrow derived MSC membrane-coated gelatin nanogels (SCMGs) showed reduced uptake by the MPS, reduced clearance by the MPS and reduced internalization by filtration organs  . As a reAmong the various camouflage strategies, cancer cell-derived biomimetic strategies have been considered as a promising option for the surface functionalization of NPs with cell membrane, due to the intrinsic homotypic aggregation properties of cancer cells and their ability to escape immunorecognition. Fang et al. previously demonstrated that cancer cell membrane-coated NPs (CCNPs) improved cancer targeting due to homotypic interaction . In partChen et al. developed biomimetic NPs by fusing a cell membrane isolated from MCF-7 human breast cancer cells to an indocyanine green (ICG)/PLGA core . Dubbed Similarly, Sun et al. developed a strategy to exploit cancer cell membrane-coated NPs for targeted chemotherapy of homotypic tumors . The autIn recent years, immune cells have also been proven to be a promising source of coating for biomimetic NPs. Specifically, our group was the first to demonstrate the beneficial properties endowed to NPs when a leukocyte-derived cell source was used. This proof-of-concept was demonstrated with the functionalization of nanoporous silicon particles with cellular membranes isolated from leukocyte cells . Dubbed To further evaluate the role cell source may have in the process of transport and targeting, we next compared syngeneic- and xenogeneic-derived cell membrane with uncoated NPs . This stA similar strategy for targeting activated endothelium was used in the design of protein-rich liposomal vesicles. Using leukocyte-derived cellular membranes, proteo-lipid vesicles  were fabricated and found to successfully incorporate more than 340 distinct proteins with a majority being associated with plasma membrane . As obseSimilar observations of anti-inflammatory effects were observed when biomimetic NPs were further functionalized with the α4β7 integrin commonly overexpressed in a T lymphocyte-subset that specializes in targeting gastrointestinal inflammation . This stSimilar strategies have also been employed in the use of peripheral blood mononuclear cells (PBMCs) as a promising source of cell membrane for NP coating. Compared to conventional NPs, PBMC-derived biomimetic nanoparticles are endowed with leukocyte properties such as immune evasion, longer blood circulation, and tumor recognition and targeting. As such, several attempts have been made to develop immune cell-derived NPs. For example, Cao et al. cloaked drug-carrying liposomes with cell membranes isolated from macrophages for targeting metastatic lungs sites in a 4T1 breast cancer mouse model. These membrane-decorated emtansine liposomes (MELs) were endowed with enhanced uptake in metastatic cells resulting in inhibitory effects on cell viability. Furthermore, MELs exhibited specific metastatic-targeting potential in vivo with anti-metastatic activity observed in a metastatic breast cancer mouse model . Based oThe tumor microenvironment is composed of tumor cells surrounded by a variety of additional cells  that participate in the cell-signaling network to create a neoplastic niche while supporting tumor growth and metastasis , 65. As Altogether, the surface modification with membrane proteins provides biomimetic NPs with the ability to decrease opsonization and prolong circulation within the body, thus facilitating NP targeting. More recently, researchers have begun to explore combining proteins extracted from multiple cell sources as a strategy to develop biomimetic NPs with multiple functions and overall greater therapeutic efficacy Fig. . DehainiAnother important player in biomimetic NP function is the protein corona (PC): a layer of biomolecules that binds on the surface of NPs when dispersed in biological fluids. Contrarily to uncoated NPs, the presence of proteins on the biomimetic NP’s surface could favor the adsorption of specific proteins over others. For example, leukocyte membrane proteins in the liposome bilayer affect the adsorption of blood soluble proteins in the corona. We studied in vivo the evolution of the PC over time and revealed that the integration of leukocyte membrane proteins into the leukosome bilayer influenced the number, amount, orientation and type of plasma proteins adsorbed by the NPs, thus affecting NP biodistribution and interaction with cells .However, despite the significant progress made in the development of biomimetic strategies, some aspects still needed further investigation to translate these NPs into a clinically-viable tool. To minimize immunoreactivity, biomimetic NPs could be ideally fabricated from the cells of the patient to be treated. However, due to the limited number of some cell subpopulations within the body, an ex vivo amplification step would be necessary upon isolation of the right cell types. Consequently, there is the need to create standard protocols to guarantee reproducibility, test batch-to-batch properties and achieve consistent systemic effects in vivo. While more studies need to be performed en route to clinical translation, experimental evidence suggests that biomimetic NPs could provide a solution to overcome major limitations and drawbacks of previous generations of NPs and could represent a promising tool in the treatment of cancer.


Head and neck cancer is the seventh most common cancer in Australia and globally. Despite the current improved treatment modalities, there is still up to 50–60% local regional recurrence and or distant metastasis. High-resolution medical imaging technologies such as PET/CT and MRI do not currently detect the early spread of tumour cells, thus limiting the potential for effective minimal residual detection and early diagnosis. Circulating tumour cells (CTCs) are a rare subset of cells that escape from the primary tumour and enter into the bloodstream to form metastatic deposits or even re-establish themselves in the primary site of the cancer. These cells are more aggressive and accumulate gene alterations by somatic mutations that are the same or even greater than the primary tumour because of additional features acquired in the circulation. The potential application of CTC in clinical use is to acquire a liquid biopsy, by taking a reliable minimally invasive venous blood sample, for cell genotyping during radiotherapy treatment to monitor the decline in CTC detectability, and mutational changes in response to radiation resistance and radiation sensitivity. Currently, very little has been published on radiation therapy, CTC, and circulating cancer stem cells (CCSCs). The prognostic value of CTC in cancer management and personalised medicine for head and neck cancer radiotherapy patients requires a deeper understanding at the cellular level, along with other advanced technologies. With this goal, this review summarises the current research of head and neck cancer CTC, CCSC and the molecular targets for personalised radiotherapy response.  The worldwide incidence of head and neck cancer is more than 600,000 cases with 350,000 deaths each year . In AustThese statistics indicate that there is an immediate need for improved therapy modalities specifically for the HNSCC patients who are at the risk of loco regional or distant metastasis. In clinical practice, it may be difficult to obtain tumour tissue from patients for gene alteration discoveries to tailor treatment. Currently, radiotherapy alone or in combination with chemo-radiotherapy has been reasonably effective for HNSCC but there is room for improvement ,14,15. HDespite the emergence of recent state-of-the-art radiotherapy modalities such as Image-Guided Radiation Therapy (IGRT), Intensity-Modulated Radiation Therapy (IMRT), Volumetric Modulated Radiation Therapy (VMRT) or Stereotactic Ablative Body Radiotherapy (SABR), there is a limitation on the precise dose delivery associated with tumour volume and on the biological effect  in deterIn 1869, the Australian physician Thomas Ashworth  discover® System  CTC test is the only method that has been approved by the FDA (Food and Drug Administration) such that it can be used as a surrogate marker of overall and progression-free survival for only few types of cancer like breast, prostate and colorectal cancers [TM and the ISET methods in 210 patients undergoing radical surgery for non-small-cell lung carcinoma (NSCLC). They concluded that both methods were good prognostic markers for CTC detection in this patient group. CTC detection methods utilised in studies investigating head and neck cancers are summarised in Solid tumour CTC isolation is commonly based on the biological properties which are enriched either by EpCAM  positive selection or CD2, CD14, CD16, CD19, CD45, CD61, CD66b and Glycophorin A negative selection of cell surface antibodies  ,30,31,32 cancers . In a co cancers  reported cancers . Khoja e cancers  compared cancers  also con cancers  explains cancers  discusse cancers  also comTo date, there have only been a few published studies investigating the impact of radiation therapy on CTCs ,69,74,75More recently, blood-based prognostic and predictive biomarkers for radiotherapy have drawn much attention and excitement in radiation oncology. In this section, we reviewed and explored all the noted uses of HNSCC circulating tumour DNA (ctDNA) for radioresistance and radiosensitivity diagnostic approaches. Primary tumour information and metastases at different sites within the same patient may have different genomic characteristics due to tumour heterogeneity . TherefocfDNA is unstable and short-lived in the bloodstream ,85,86,87Protein-based circulating biomarkers are specific to one cancer type, and it is not feasible to compare and generalise this to all cancer types . On the Next-generation sequencing technology platforms for ctDNA analysis was developed 20 years ago ,107,108 Measuring Circulating Epstein Barr virus (EBV) DNA and human papilloma virus (HPV) DNA in HNSCC patients can be used as a biomarker for before and after radiotherapy and chemo-radiotherapy response. Successful radiotherapy treatment should decrease the level of circulating EBV and HPV DNA ,108,112.Currently, only clinical radiotherapy imaging data, as indicated in CTC culturing for functional studies from cancer patients is logistically challenging. Currently, for understanding CTC, CCSC biology and tumour heterogeneity, all race cell lines or preclinical models for developing new efficient novel drugs are not commercially available . FunctioCancer stem cells (CSCs) are cells possessing self-renewal properties with high metastatic potential and resistance to therapies. The major reason for treatment failure in anticancer therapy modalities is due to the presence of CSCs. CTCs expressing stem cell markers in the circulation are known as circulating cancer stem cells (CCSCs) and are highly tumorigenic compared to tissue CSCs . CSCs anWith regard to HNSCC HPV status, HPV-positive patients responded to radiation treatment better when compared to HPV-negative patients. Vlashi et al.  tested tIn summarising the literature, the CCSC findings from several studies reveal that CTCs from head and neck, lung, breast and prostate cancer can be cultured as spheroids or organoids for more than 6 months, and these CTCs possess self-renewal cancer stem cell properties and exhibit a radioresistant and chemoresistant phenotype with a high metastatic potential ,128,129.+ CSC-like cell population possessed dose-dependent resistance to cisplatin in the peripheral blood circulation as do CCSC [The authors suggested the CD44 do CCSC . The sam do CCSC . Recentl do CCSC . Interes do CCSC  reported do CCSC . In a co do CCSC . Similar do CCSC ,135,136.Kantara C et al. also developed a novel diagnostic assay, expressing CSC markers (DCLK1/LGR5) with CD44/Annexin A2 for detecting colon cancer CCSCs in the blood of colonic adenocarcinomas patients . SimilarManagement of head and neck cancers to date has relied on the conventional treatments of surgery, radiotherapy and chemotherapy. Initiating treatment of this group of cancers by a molecular approach is innovative, and currently there is limited evidence available regarding the effects of CTC concentration during radiotherapy treatment to gauge responses and modify radiation doses. The CTC genes regulating the radiotherapy kinetics  have not been identified to date with particular applications of radioresistance and radiosensitivity. Furthermore, CTC gene expression changes after radiotherapy, and chemotherapy management is still in the infancy stages of discovery. At present, much of the work done has been focused on signalling pathways. Deregulated signalling pathways contribute to oncogenic cellular transformation and resistance. Several gene therapy strategies with different vector delivery mechanisms are being investigated in clinical trials for HNSCC in the UK ,142,143.In a collaborative study in 2009 between the Moffitt Cancer Centre, Research Institute USA and the Netherlands Cancer Institute, the multigene expression model for predicting the intrinsic tumour radiosensitivity and treatment response in patients was developed . SubsequII) is a cytotoxic drug currently used in the treatment of several cancers and is effective through causing DNA damage or apoptosis of tumour cells. The usage of cisplatin as a radiation sensitiser in the treatment of advanced HNSCCs are summarised in phase II clinical trials [Gemmill et al. and Wagner et al. ,160 critl trials ,167,168.l trials . An ongol trials . Similarl trials . In a Phl trials . NimorazWnt signalling plays a significant role in radio resistance. To our knowledge, the role of radio resistance and the Wnt signalling mechanism is not yet elucidated in any of the cancer circulating tumour cells and its associated circulating cancer stem cells. There is, however, limited literature regarding Wnt signalling in circulating tumour cells. Some data exist surrounding the non-canonical CTC gene signatures and WNT2 candidate gene in pancreatic CTC, Wnt expression contributing to antiandrogen resistance by single cell analysis RNA-seq in prostate CTCs ,173,174.Since 1997, our research group has extensively published on Wnt signalling and the role of secreted frizzled related protein (sFRP4) and its associated domains, cysteine rich domain (CRD) and netrin-like domain (NLD) as an antiangiogenic protein in various cancers such as brain, breast, ovary, prostate, mesothelioma, including HNSCC CSCs and now recently in reproductive CSCs of breast, prostate and ovary ,186,187.Based on our published data, we hypothesise the sFRP4 and its associated domains CRD and NLD will modulate the WNT 3a gene expression-induced radioresistance in HNSCC CTC and CCSCs and could reverse the effect of radioresistance. By diligently moving forward one step at a time, this potent and selective inhibitor may assist drug discovery and has potential to reverse the effect of radioresistance by targeting human HNSCC-derived CTCs and CCSCs resulting in a novel radiotherapy breakthrough for anticancer therapeutic modalities in the clinic ,184,188.From a clinical perspective, a high level of nuclear β-catenin accumulation was detected in colorectal cancer, HNSCC ,190, ionc-Met is frequently altered in 90% of HNSCC tumours by mutation and overexpression contributing to HGF/cMet signalling ,198,199.This review article provides mounting evidence and scope for using CTC counts, CTC-derived CCSC and ctDNA as a prognostic and predictive marker for current head and neck individualised radiotherapy and chemo-radiotherapy response. Tracking CTC counts during radiotherapy treatment will measure the outcome and disease resistance leading to recurrence especially in loco regionally advanced disease with a significant potential for clinical application. In future, it may also set the stage for developing CTC radiosensitive index by GARD in the clinical setting for tailored radiotherapy dose prescription clinical practice. Today cost effective NGS and other sequencing platforms can also guide the radiotherapy genomic alterations response. Our group is currently developing CTC, circulating cancer stem cells and imaging guided genetic information (model) framework for radiotherapy resistance and sensitivity in HNSCC for clinical practice. We are confident that the proposed research will also contribute to the development of new therapeutic strategies for radiotherapy precision medicine. Identification of radiotherapy/chemo-radiotherapy relapsed HNSCC CTC and CCSC subpopulation and associated genetic material will be a valuable clinical tool to improve the current treatment approach for this disease. Developing a less invasive CTC, CCSC as a prognostic and predictive marker will be of added advantage in clinical practice for sequential radiotherapy and chemo-radiotherapy assessment. Furthermore, investigating WNTs, c-Met, PI3K, AKT/mTOR, CCSC epithelial markers and stem cell markers in radiotherapy HNSCC CTCs has the potential to generate future radio-sensitisation modalities. Globally, current drug discovery is an expensive and time-consuming process with high failure rates in patients. Future studies investigating CTCs on early and advanced cancer models offer a less invasive approach compared to tissue biopsies and can facilitate further understanding of tumour heterogeneity. The aim is to tailor treatment to individual patient requirements based on information gained by conducting chemo-radiation testing on their own CTC/CCSC organoids. This would then identify the most suitable FDA-approved chemo-radiation regimen for their particular cancer. Using the advanced CTC/CCSC organoid models, resistance and sensitivity rate can be predicted for therapy response prior to administering the therapy to the patients, avoiding unwanted toxicity burden. Patient CTC organoids approach in the real world is faster and less expensive with more control than animal models. Our goal is to introduce CTC/CCSC as companion diagnostics and clinical intervention for current available treatment prediction. Identifying and targeting specifically the CSC in CTC using surface markers and combining with radiotherapy will be major advancements in the field of cancer research. Introducing such robust platform discoveries and translating it to human research in radiotherapy treatment will help resolve the current challenges of potentially incurable cancer recurrence.