Introduction: Multi-disease community health campaigns can be effective for population-wide HIV testing in a research setting (SEARCH: NCT01864603). We sought to evaluate feasibility and uptake of a community-led health campaign (CLHC) planned and implemented by village leaders and local clinic workers in Uganda.Methods: Over five months in 2014, locally elected village leaders and Ministry of Health (MoH) clinic staff in a rural parish in Uganda planned a census followed by a CLHC, after training by two SEARCH trial consultants and by leaders from a neighbouring parish that had previously participated in a SEARCH health campaign. We defined feasibility as: (1) elected leaders’ participation in training and implementation of pre-campaign census and mobilization activities; (2) implementation of all campaign activities by MoH-funded, local clinic staff; and (3) community participation in the campaign, including point-of-care screening for HIV, malaria, hypertension and diabetes, and same-day referral for male circumcision and family planning (FP). Costing of all salaries and supplies was conducted.Results: Elected leaders from all eight villages in the parish participated in CLHC training. They and local clinic staff met monthly to select and plan CLHC services. Village leaders then leveraged existing volunteer health teams to perform a door-to-door census, enumerating 5,202 parish residents over 2 weeks. 2,753 (53%) residents participated in the 6-day CLHC. Of 1,584 adult participants, 1,474 (93%) tested for HIV: 105/1,474 (7.1%) tested HIV positive. 27%  of participants reported fever and underwent malaria rapid diagnostic testing: 5.3% (40/751) tested positive. Among adults screened, 19%  were hypertensive, and 3% (18/637) had a random blood sugar >11.1 mmol/L. Of 805 men and boys (>10 years), 91 (11%) accepted same-day clinic referral and underwent medical circumcision. Of 900 women offered same-day long-term FP referrals, 25 accepted. The CLHC cost, including census, mobilization and testing services, was $23,597 ($8.57/participant).onclusions: Elected village leaders successfully planned and conducted a 6-day multi-disease health campaign with service provision by local clinic staff that reached over half of a rural Ugandan community. These data suggest it is feasible for local leaders and clinics to adopt a multi-disease health campaign approach to scale-up HIV testing in rural Africa.C Increasing HIV testing coverage remains a challenge for most of sub-Saharan Africa, where less than half of HIV-infected persons are estimated to know their HIV status . To achiMulti-disease community health campaigns are an effective way to rapidly increase HIV testing coverage in rural east Africa –4, as deIn Uganda, Ministry of Health (MoH) policy recommends health promotion through empowerment of local communities to participate in decision-making and planning for local health service provision ,9. On a Rwebishekye Parish in Mbarara District in south-western Uganda is a rural agricultural community composed of eight villages and an estimated 6,400 residents based on 2002 Uganda National Census projections for 2012 . BwizibwWe defined feasibility of the community-led health campaign as: (1) elected leaders’ acceptance and participation in campaign training, and implementation of pre-campaign census and mobilization activities; (2) implementation of all campaign activities by local MoH-funded clinic staff. This definition of feasibility focuses on aspects of health campaign implementation by locally elected leaders , and by local clinic staff  that have previously been staffed by research assistants in the SEARCH trial, in order to provide data on how to transition from research-led to community-led campaigns.We defined uptake as census-enumerated resident participation in campaign activities, including point-of-care screening for HIV, malaria, hypertension and diabetes, and same-day referral for safe male circumcision and family planning services to parish residents.Elected village leaders and local MoH leadership were approached by two consultants from the SEARCH Trial and from elected village leaders from a neighbouring parish that had previously participated in a SEARCH health campaign, and invited to receive training over three days on implementation of community census and pre-campaign mobilization activities. Training included review of how health campaigns are organized (including participant “flow” through the campaign stations), how to create and use census registers, and how to record campaign participant information. Elected leaders held three meetings with local MoH-funded clinic staff to plan, select and agree on campaign health services based on local health priorities.Elected leaders designed and implemented community mobilization activities, including community meetings, announcements at public gatherings, and the distribution of posters and flyers in the 5 months before the campaign from April to August 2014. Village leaders selected three well known and centrally located sites across the parish for the campaigns, so as to maximize participation and reduce participants’ transport costs to the campaign.Over 2 weeks in August 2014, elected village leaders worked with existing local volunteer health teams  to perform door-to-door enumeration of all residents living in their assigned villages. If no adult resident was home during an initial census visit, two additional visit attempts were made , in an attempt to reach all residents. The census allowed the leaders to obtain an estimate of the baseline population size and of testing coverage at the campaign. Census workers used a simple register to record basic information about household members such as age, sex, marital status, and occupation. Identification numbers were created and assigned to all residents during census enumeration. Campaign workers then used resident name, gender, age, and village to identify and verify campaign participation.Elected village leaders and VHTs worked directly with MoH clinic staff to choose the services provided at the CLHC based on local health priorities and local clinic resources, and designed the CLHC in consultation with advisors from the SEARCH trial and leaders in the neighbouring community of Kakyerere Parish who had experience in conducting CHCs, as previously described ,7. LocalElected leaders and MoH clinic staff selected the following services for the CLHC: screening for HIV, hypertension, diabetes and malaria, male condom distribution, referral for medical circumcision for men, and family planning services for women. Both medical male circumcision and family planning referral services were not initially presented as options by the SEARCH trial consultants, but rather added on the suggestion of MoH clinic staff during planning meetings. Over a period of six consecutive days for 8–10 h/day), MoH clinic staff at the campaign performed rapid point-of-care screening for HIV following Uganda MoH guidelines –10 h/day and malaServices at the CL-CH were provided in the following order: after initial registration, participants were first directed to a “Vital signs station” for measurement of blood pressure, before accessing the CL-HC laboratory for finger-prick blood testing . Additional services for which referrals were made , took place during one-on-one post-test counselling, prior to the CL-HC exit station. At the exit station, Vitamin A supplementation and de-worming treatment services were provided, male condoms distributed to adults, and a raffle took place for those who completed all campaign activities, in which campaign participants could win prizes. The total value of all raffle prizes distributed was US $500.Measures of prevalence of each disease were estimated using the sample proportion with the total number of participants screened as the denominator. Stata version 11 software  was used for analysis.All census and campaign expenses were identified through extraction of cost information from expenditure records and study logs, supplemented by interviews with SEARCH consultants and MoH clinic staff that delivered the services. For the elected leaders and volunteer health teams, we calculated the cost of six days of daily transport reimbursements to reach CL-HC sites. For the campaign, the costing methods focused on resources utilized and economic costs, rather than out-of-pocket costs. We classified the resources under four main categories: personnel (including fringe benefits), recurring supplies and goods, recurring services , and capital goods and equipment. Personnel costs included a daily per diem for MoH workers during the six campaign days to cover off-site work. Costs of capital items were amortized on a straight-line basis over three years for lab equipment and 5 years for furniture, vehicles and computers, assuming no salvage value. The other activity costs also included overhead and administrative costs, training (including consultant costs for each day of training), community mobilization (including the cost of poster/flyer printing and distribution), transportation (including transport reimbursements during the census and campaign days), counselling, and laboratory-testing costs for all diseases screened. All costs were converted to US dollars based on the 2014 exchange rate of 2,463 Ugandan shillings per US dollar.For a scaled-up replication model, we projected costs using MoH facilities, capital goods and government staff average salaries, without SEARCH trial support. Modified costs include using existing school or health centre facilities infrastructure rather than SEARCH-trial infrastructure ; using MoH salaried coordinators and laboratory technicians; excluding SEARCH-supported expenses .The Makerere University School of Medicine Research and Ethics Committee and the Ugandan National Council on Science and Technology (Uganda), and the University of California, San Francisco Committee on Human Research approved the consent procedures and the study. All participants, or their parent/guardian, provided verbal informed consent in their preferred language. Children from 13–17 years could provide verbal consent if a parent/guardian was not present, whereas children <13 years old could not participate without an assenting parent/guardian present, in accordance with Uganda Ministry of Health guidelines .All locally elected leaders from the eight villages comprising Rwebishekye parish participated in the training and planning for the census, mobilization activities and the health campaign see . ThroughOf 29 MoH-funded clinic healthcare workers at Bwizibwera Health Centre, 20 participated in campaign service provision throughout the CLHC, while the other nine remained at the health facility to perform ongoing, routine clinic activities and to accept referrals from the CL-HC. MoH clinic healthcare workers provided all healthcare service delivery, including point-of-care laboratory testing, and were present for the full duration of activities on all six days of the CLHC see .Specific feasibility challenges when working with locally elected leaders, VHTs and MoH health clinic workers included low literacy levels, which made name matching of census lists and campaign participants challenging due to misspellings, and generated occasional data entry errors when recording screening results. In addition, many of the VHTs and MoH workers anticipated additional payments for their work apart from the daily transport reimbursements and per diems (described above), respectively, on campaign days.Out of an estimated 6,400 residents in the parish based on national census projections, local leaders enumerated a total of 5,202 residents in two weeks; 78% of the projected population. The census enumerated 2,162 children (<15 years), and 3,016 adults (≥15 years); 24 residents were enumerated without age information. Overall, 1,205 households were enumerated with an average household size of 3 persons (range: 1–15 persons/household).Over six campaign days in September 2014, 2,753 (53%) residents participated in the CLHC, including 48%  of enumerated children and 53%  of adults; for 132 campaign participants’ age was not recorded. Among adult (≥15 years) campaign participants, 597/1,584 (38%) were men , and 1,4Out of 900 women (≥18 years) participating in family planning education at the CLHC, 25 (2.8%) accepted same-day referral to the local clinic for long-term family planning services and received either an IUD (n = 2) or an implant (n = 23); 20/25 (80%) women who accepted FP interventions were <32 years of age. Of 805 men and boys >10 years old evaluated for safe and voluntary, medical male circumcision, 91 (11%) accepted same-day referral to the local clinic for circumcision. 107/2,753 (3%) participants presented for urgent care evaluation and received treatment for their presenting complaints at the CLHC: those that required care that could not be addressed at the CLHC were referred to the health centre for further management.The total operating cost of providing the census, mobilization activities, and all campaign services, including SEARCH consultation , was $23,596.56 (2014 US dollars); an average of $8.57 per participant . SEARCH In this study, a health campaign planned by local village leaders and implemented by local clinic healthcare workers was able to reach over half of residents in a rural Ugandan community of over 5,000 persons within one month of planning and six days of implementation. Multi-disease health campaigns have been successfully implemented in sub-Saharan Africa and represent an effective strategy for rapid population-wide scale-up of HIV testing in rural, resource-limited settings –5. HowevCommunity involvement in health campaign service delivery in resource-limited settings is likely to have a positive impact on health-seeking practices by local residents, as a community-led approach allows local leaders and clinic staff to exercise choice in selecting services that match residents’ health priorities. The importance of community involvement among other factors has also been demonstrated in community-based health campaigns that have not included HIV testing, including vaccination campaigns and bed net distribution, with or without the bundling of health services –14. In RTo our knowledge, our study is among the first to demonstrate the feasibility of conducting a rapid census and multi-disease health campaign, led and implemented by locally elected leaders and VHTs and local MoH clinic staff, respectively. Conducting a pre-campaign census serves a dual purpose of mobilizing residents to participate in the campaign while publicly demonstrating local leader support for the campaign, and providing a direct measure of the proportion of the community covered by campaign activities. This latter measure of coverage may serve as an indicator of the campaign’s success, thereby providing direct feedback to locally elected leaders and MoH clinic healthcare workers on which demographics they are failing to reach in their communities. The feasibility of a local census also suggests that a community-led hybrid approach to out-of-facility HIV testing, in which home-based testing of campaign non-participants is implemented after the campaign to maximize community coverage , may be The cost estimate of this multi-disease community-led campaign (including census enumeration and SEARCH support) of $8.57 per participant (2014 USD), is comparable to other community-based HIV testing approaches ,19, and This study has several limitations. First, our study may have overestimated campaign coverage if some residents were missed during the census. Additionally, name matching of the enumerated villagers and CLHC attendees was challenging due to variable spelling of names and multiple names provided by individuals. Biometric fingerprint matching is a more accurate method of identification and could be added into future CLHCs to address this challenge . Second,In summary, this community-led health campaign demonstrates the feasibility of having community leaders and health facilities plan and conduct a rapid census enumeration, mobilization activities, and a health campaign, with consultative support. With this community-led approach, over half of community residents participated in testing for HIV, while accessing other preventive health and treatment services. These data suggest that a community-led approach to multi-disease health campaigns is feasible, and could serve as a platform for rapid scale-up of out-of-facility HIV testing in a rural African setting.


Breastfeeding is associated with reduced mortality in children aged less than 5 years. We conducted a systematic review and meta-analysis (registered as PROSPERO 2015: CRD42015019105) to examine the effectiveness of community-based peer support for mothers on their breastfeeding practices as compared to mothers who have not received such a support.We searched for evidence regarding community-based peer support for mothers in databases, such as PubMed/MEDLINE, the Cochrane Library, CINAHL, Web of Science, SocINDEX, and PsycINFO. We selected three outcome variables for breastfeeding practices, namely, exclusive breastfeeding duration, breastfeeding within the first hour of life, and prelacteal feeding. We conducted meta-analyses of the included randomized controlled trials and quasi-experimental studies.For our review, we selected 47 articles for synthesis out of 1,855 retrieved articles. In low- and middle-income countries, compared to usual care, community-based peer support increased exclusive breastfeeding at 3 months , at 5 months  and at 6 months . In high-income countries, compared to usual care, peer support increased exclusive breastfeeding at 3 months . In low- and middle-income countries, compared to usual care, peer support increased the initiation of breastfeeding within the first hour of life  and decreased the risk of prelacteal feeding .Community-based peer support for mothers is effective in increasing the duration of exclusive breastfeeding, particularly for infants aged 3–6 months in low- and middle-income countries. Such support also encourages mothers to initiate breastfeeding early and prevents newborn prelacteal feeding. Appropriate breastfeeding practices improve child survival, health, and development . GloballEven though these recommendations were issued more than 25 years ago, breastfeeding rates still remain far below set targets in many countries . For exaEvidence is available on how effective peer support interventions for mothers (one-on-one or in a group) can be in improving breastfeeding practices, but the results remain inconsistent , 11. We We developed and followed a standard systematic review protocol (systematic review registration-PROSPERO 2015: CRD42015019105) in accordance with the PRISMA statement S1 TableS1 Table.We reviewed studies involving mothers of children less than five years of age.Studies were eligible if they focused on one-on-one and/or group peer support for mothers, including peer nutrition counseling, shared decision making, grandmothers/elders-to-mother nutrition counseling. We also included studies that had nutrition-focused participatory interventions involving mothers themselves as key drivers. We excluded studies that included top-down nutrition interventions, such as the distribution of ready-to-use therapeutic foods, supplemental blanket feeding, and cash transfer.The primary outcome of the original study protocol was the child's nutritional status , and the secondary outcome was child feeding practices (including breastfeeding and other feeding practices). However, we did not find a sufficient number of eligible studies that analyzed the role of community-based interventions on child nutritional status and feeding outcomes, such as complementary feeding, feeding frequency, and dietary diversity. Therefore, we focused on breastfeeding practices. We utilized WHO definition of EBF and defined EBF duration as the period when an infant receives only breast milk without any other liquids or solids, including water and expressed breast milk. We included breastfeeding initiation within the first hour of life and prelacteal feeding as post-hoc additional outcomes. We defined prelacteal feeding as any food or fluid provided to a newborn before initiation of breastfeeding.We selected studies that included randomized controlled trials (RCTs), quasi-experimental, cohort, cross-sectional, and other comparative observational studies.We conducted the search process in six medical databases. These were as follows. PubMed/MEDLINE, the Cochrane Library, CINAHL, Web of Science, SocINDEX, and PsycINFO. We used the following search strategy for the PubMed/Medline database:  OR groups, women's[MeSH Terms]) OR club, mothers'[MeSH Terms]) OR mothers[MeSH Terms]) OR clubs, mothers[MeSH Terms]) OR mother's group) OR mother's groups)) AND (((((((((empowerment[MeSH Terms]) OR community based participatory research[MeSH Terms]) OR participatory research, community based[MeSH Terms]) OR participatory intervention) OR peer groups[MeSH Terms]) OR peer group[MeSH Terms]) OR counseling[MeSH Terms]) OR peer counseling) OR shared decision making[MeSH Terms])) AND  OR sciences, child nutritional[MeSH Terms]) OR nutritional index[MeSH Terms]) OR anthropometry[MeSH Terms]) OR nutrition) OR feeding pattern[MeSH Terms]) OR feeding behavior[MeSH Terms]) OR feeding behaviors[MeSH Terms]) OR feeding practice) OR complementary feeding[MeSH Terms]) OR breastfeeding[MeSH Terms]) AND (("1978"[Date—Publication]: "2015"[Date—Publication])). Then we used similar keywords to search in the other databases selected.We also searched international organization databases such as the World Bank, UNICEF, and the WHO databases. We used the cited references of retrieved articles to hand search. We limited all the evidence to abstracts published in the English language between 1978 to the end of March 2015. The year 1978 was selected because the Alma Ata declaration was made in this year, and it emphasized community participation as an important primary health care component.We allocated the searched and selected articles equally among the four review teams. Then, two researchers in each review team independently performed the eligibility assessment in a blinded standardized manner.The two researchers in each review team independently assessed the risk of bias for the studies included. We used the risk of bias assessment tool developed by Cochrane collaboration for assessing RCTs and quasi-experimental studies . For obsThe two researchers in each review team independently extracted the data and entered it in a standardized data extraction matrix. The following information was retrieved during data extraction: study location, design, participants, type of intervention, training of the mothers, the presence of professional help, the comparison group, and reported outcomes of interest.A meta-analysis of RCTs and quasi-experimental studies using Review Manager (Revman) 5.1 software was conducted. We used a random effect model of meta-analysis throughout and presented results as risk ratios (RRs) with their 95% confidence intervals (CI).2 statistic. We also performed sensitivity analysis, where appropriate, to evaluate whether pooled effect sizes were robust across the components of risk of bias.We conducted the meta-analyses by stratifying the studies into LMICs and high-income countries as defined by the World Bank . We alsoWe included 22 studies in the meta-analyses of different outcomes. We excluded two studies that assessed multifaceted interventions. Such multifaceted interventions make it difficult to isolate and attribute the effect of peer support for mothers , 18. We This study was a review and dealt only with data published in the selected studies. We had no direct access to the original data used in any of those studies. Therefore, no ethical approval was warranted for this review.We noted differences in the inclusion criteria of the study populations. The enrollment period for mothers varied among the studies, from any time during the antenatal period, either the first, second or third trimester of pregnancy to the postpartum period. Two studies exclusively included mothers who had low birth-weight infants , 24.We also noted differences in the type of interventions, which included either one-on-one counseling or mothers' group meetings. Of 47 studies, 38 focused on one-on-one counseling with the remaining nine focusing on mothers' group meetings , 25–28. We noted differences in the risk of bias among the studies  Tables. We included14 studies in the meta-analysis to measure the effect of community-based peer support for mothers on EBF duration in LMICs . Among t2 = 68%).We included two studies in this subgroup analysis , 39 whic2 = 71%).We included four studies in this subgroup analysis , 21, 39 2 = 98%).We included three studies in this subgroup analysis , 24, 40 2 = 30%).We included two studies in this subgroup analysis , 32 whic2 = 94%).We included 10 studies in this analysis , 39–41 wWe included eight studies in this analysis to measure the effect of community-based peer support for mothers on EBF duration in high-income countries . Among t2 = 90%).We included two studies in this subgroup analysis , 45 whic2 = 63%).We included five studies in this subgroup analysis , 43, 46 2 = 81%).We included three studies in this subgroup analysis , 44, 45 2 = 73%).We included four studies in this subgroup analysis –45, 47 w2 = 22%).We included two studies in this subgroup analysis , 47 whic2 = 98%).We included four studies in this analysis to measure the effect of community-based peer support for mothers on breastfeeding initiation within the first hour of life , 32, 41.2 = 56%).We included two studies in this analysis to measure the effect of community-based peer support for mothers on prelacteal feeding of infants , 32. BotWe conducted subgroup analyses by the type of intervention (one-on-one or group peer support) for the outcome of EBF until 6 months. We included 12 studies in this analysis. Among them, two were quasi-experimental studies  and the 2 = 90%).The eight studies in this subgroup included 6,254 mothers. The pooled relative risk of continuing EBF until 6 months was 3.24 (95% CI: 2.04–5.14) for the intervention group versus the control . This es2 = 96%).The four studies in this subgroup included 18,849 mothers. The pooled relative risk of continuing EBF until 6 months was 2.77 (95% CI: 1.71–4.51) for the intervention group versus the control . This esWe conducted a sensitivity analyses to observe the effect of studies that had high risk of bias. We did this by comparing before- and after- meta-analysis results of the studies. However, we did not find any substantial differences or any publication biases.This systematic review highlighted four major findings on the effectiveness of community-based peer support for mothers on their breastfeeding practices. First, community-based peer support for mothers can improve EBF duration. Moreover, mothers in LMICs continued EBF for a longer period than mothers in high-income countries. Second, such interventions can improve breastfeeding initiation within the first hour of life in LMICs. Finally, community-based peer support can reduce prelacteal feeding in LMICs.Community-based peer support for mothers significantly increased EBF duration among mothers in both LMICs and high-income countries. In LMICs, mothers who received such support exclusively breastfed their infants until 3, 5 or 6 months compared to those who did not have such support. They even continued EBF until later months as suggested by the higher risk ratios in meta-analyses results at 5 and 6 months. In high-income countries, mothers who received such support exclusively breastfed their infants until 3 months compared to those without such support. We did not find any significant results proving effectiveness of peer support for EBF until the fifth and sixth months in high-income countries.Mothers who received peer support in LMICs tended to exclusively breastfeed their infants for a longer period than such mothers in high-income countries. A separate systematic review also found that peer support had greater effect on EBF in LMICs than in high-income countries . One posMothers who received peer support in LMICs were more likely to initiate breastfeeding within the first hour of life than those who did not have such support. Moreover, peer support also decreased the risk of prelacteal feeding of newborns in LMICs. A separate systematic review also reported peer support as an effective intervention to promote breastfeeding initiation among low-income group mothers .Our findings should be interpreted in line with the following limitations. First, we found differences in study populations, type of interventions, training methods, and outcome measurement methods, all of which may modify the effect of the interventions. This may also explain the source of substantial heterogeneity in some of our meta-analysis results. We addressed this by conducting subgroup analyses based on EBF duration and type of interventions (one-on-one or group peer support). Some of the subgroup analyses at different EBF follow-up time points were based on relatively smaller samples and may not have had the power to identify significant effects. We included quasi-experimental studies in the meta-analysis, which may have increased the risk of bias.We were not able to assess the effect of community-based peer support for mothers when it was integrated into a multifaceted intervention. Only two studies included a multifaceted intervention. In the first study, the packaged intervention was the Integrated Management of Childhood Illness (IMCI) that significantly increased EBF duration . The secDespite these limitations, this study is the first systematic review and meta-analysis to report the effectiveness of community-based peer support for mothers for different EBF durations ranging from 1 month to 6 months. Our findings may help policy makers to design low-cost and sustainable strategies to improve breastfeeding practices in locations where effective programming is lacking.Community-based peer support is effective in increasing EBF duration among mothers. Therefore, the mothers in LMICs continue EBF for much longer periods than the mothers in high-income countries. Such community-based peer support also enabled mothers to initiate breastfeeding early and avoid prelacteal feeding of newborns in LMICs. Moreover, mothers are more likely to exclusively breastfeed when they receive peer support one-on-one or through a mother’s group. Future studies are needed to explore sources of heterogeneity in such estimates and also examine the effect of a multifaceted intervention on breastfeeding practices.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Using pharmacological and genetic alterations, we mechanistically demonstrate the critical importance of BDNF-TrkB signaling in the tumor microenvironment. These investigations further support the rationale for BDNF/TRKB targeted therapy against in the treatment of HNSCC.It has long been known that the tumor microenvironment contributes to the proliferation and survival of neoplasms through the constant interaction with the stromal and immune compartments. In this investigation, we explored the role of cancer-associated fibroblasts (CAFs) in the regulation of the tumor microenvironment in head and neck squamous cell carcinoma (HNSCC) though a complex intercellular BDNF-TrkB signaling system. Our studies show that conditioned media derived from patient-derived CAFs promoted HNSCC cell proliferation, in vitro cell migration, cell invasion and chemotherapy resistance, compared to normal fibroblasts. Furthermore, examination of the  Although metastasis to the regional nodes represents a coordinated cellular and molecular ballet of distinct cell populations in the tumor microenvironment, the fundamental contributions of these protagonists to lymphatic metastasis are still poorly understood.The involvement of regional lymph nodes is the primary mode of metastasis for most solid tumors, a clinical phenotype that has a profound impact on therapeutic strategies and survival in cancer patients. For head and neck squamous cell carcinoma (HNSCC), lymphatic metastasis occurs in over 50% of all patients, and its presence portends for the development of distant metastasis and adverse prognosis. Although improvements have been achieved in surgical techniques, radiation therapy and systemic treatments, the overall five-year survival for patients with lymphatic metastasis remains unaltered. While molecular-targeted agents have demonstrated anti-cancer efficacy in pre-clinical models and clinical trials for HNSCC –10. While the elucidation of some of these micro-environmental cues has shed light on how bi-directional communication between CAF and tumor cells impacts malignant progression, whether CAFs are instrumental in vascular and lymphatic metastasis has not been fully explored to date.The oncogenic milieu is comprised not only of primary tumor cells, but also endothelial and inflammatory cells, that have been shown to promote carcinogenesis, tumor growth and metastasis. Yet, the major cellular component of the stromal architecture is the fibroblastic compartment, which has emerged as a functional contributor to the tumor microenvironment. Termed cancer-associated fibroblasts (CAFs), these mesenchymal cells have been shown to mediate diverse pro-oncogenic processes, including malignant transformation, inflammation, angiogenesis and invasion. Clinically, the extent of fibroblastic response is has been linked to survival outcomes among patients with various cancers, including HNSCC. Although recent evidence implicates CAF-controlled signaling pathways in modulating the tumor microenvironment, the molecular mechanisms underlying these CAF-mediated processes are incompletely understood –13, and several lines of evidence demonstrate the role of the brain-derived neurotrophic factor (BDNF) and its cognate receptor, TrkB, in various cancers –19. Previous studies have revealed roles for BDNF-TrkB in malignant transformation, tumor progression, metastasis, angiogenesis, and therapy resistance –27. These studies suggest that the BDNF-TrkB axis may be a critical component of multi-step tumor progression in carcinomas, although the precise mechanisms for its contribution are incompletely understood. While in certain disease models, tumor-derived BDNF instigates abnormal intracellular pathway upregulation in an autocrine and paracrine manner, whether CAF-derived BDNF ligand contributes to a putative CAF-tumor synapse in potentiating lymphovascular invasion has not been explored to date.Investigation over the last twenty years has implicated neurotrophin signaling in multiple biological processes in vitro and in vivo. Further, CAF-derived BDNF contributes mechanistically to the development of a pro-invasive and pro-angiogenic metastatic milieu that promotes aggressive tumor behavior and adverse outcomes among patients with squamous cell carcinoma.In this study, we demonstrate the important role of human tumor-derived CAFs in mediating tumor metastasis that is mechanistically driven by the BDNF-TrkB signaling pathway. Specifically, using model systems of squamous cell carcinoma, we show that CAFs directly control the biological behavior of aggressive malignancies , 29. The OSC19-Luc cell line is a stable transfectant constitutively expressing luciferase and GFP . Normal human fibroblasts (Detroit 551) were purchased from ATCC. Recombinant human BDNF and EGF were obtained from Peprotech . The following antibodies were utilized: TrkA , TrkB (sc-8316), TrkC (sc-117), NGF (sc-548), BDNF (sc-546), vimentin (sc-51719), GAPDH, MAPK , phospho-MAPK (9101), STAT3 (9132), phospho-STAT3 (9145), AKT (9272), phospho-AKTser473 (3787), Twist (4119), E-cadherin (4065), N-cadherin (4061), β-catenin (9562), PCNA (2586), Slug, Snail and LYVE-1.The head and neck squamous cell carcinoma cell lines OSC19, UMSCC1, Tu138, FADU, UMSCC-22A cells were maintained as described previously 2-humidified atmosphere. CAFs grew around the explants and were cultured for approximately 2–3 weeks. Pieces of tissue fragments were removed, and cells were trypsinized and seeded at 70% to 90% confluence and cultured in complete medium. When cells reached 95% to 100% confluence (~1.7–2.0 × 104 cells/cm2), they were washed with PBS, incubated with serum-free medium for 1 hour, washed again, and incubated for 24 hours in serum-free medium.Cancer-associated fibroblasts were harvested from human HNSCC tumor tissues by using an explant technique. All procedures were performed under an IRB-approved protocol. Briefly, ex vivo biopsies were taken in a sterile manner from surgically resected tumors within 30 minutes of tumor resection. Tissues were cut with scissors and scalpel into smaller pieces of 2–3 mm and planted on a tissue culture dish in fibroblast growth medium-2 (Lonza) containing 1% penicillin/streptomycin (Invitrogen) at 37°C in a 5% COSmall interfering RNA targeting BDNF, GAPDH (Ambion) and AKT (Sigma) were transfected into cells according to the manufacturer’s protocol. Confirmation of target gene downregulation was confirmed after 48 hours. Short-hairpin RNA (shRNA) constructs targeting TrkB or BDNF were purchased from Origene (Rockville) and were introduced into cells via retroviral infection according to the manufacturer’s protocol and selected with puromycin (1 μg/ml).ImagePro  and Prism . Densitometry data were analyzed by utilizing either conventional Student’s t test or ANOVA followed by post hoc comparisons based upon modified Newman-Keuls-Student procedure, where appropriate. Results are reported as mean +/− SEM. A p value of < 0.05 was considered significant and all are two-tailed.Cells were grown to 80% confluency, washed with PBS and lysed for 30 minutes on ice . SDS-PAGE analysis was performed, and membranes were incubated overnight at 4° with antibodies directed against the indicated proteins. Membranes were washed, incubated with the appropriate secondary antibodies and exposed to Lumi-Light Western Blotting Substrate kit . Images were analyzed with max® ImmunoAssay System using the manufacturers’ instructions . Samples were measured in triplicate, and the mean value was used for analysis. Protein levels were analyzed by the ANOVA test followed by post hoc comparisons based upon modified Newman-Keuls-Student procedure. Results are reported as mean +/− SEM. A p value of < 0.05 was considered significant and all are two-tailed.Supernatants were collected from treated cells after 24 hours, filtered, centrifuged and stored at −80°. Levels of secreted BDNF were measured in the cultured supernatant with BDNF E5 and treated the next day with various inhibitors. After 24 hours post-treatment both detached and attached cells were harvested and washed once in PBS. Cells were centrifuged at 200g for 5 minutes and the pellet was resuspended in annexin binding buffer and incubated in annexin V for 15 minutes in the dark according to the recommendation of the manufacturer . Propidium iodide flow cytometric analysis were performed on Gallios .Cells were seeded in 6-well plates at a density of 3×10. Differences between groups were analyzed by utilizing either conventional Student’s t test or ANOVA followed by post hoc comparisons based upon Bonferroni’s Multiple Comparison Test, where appropriate. Results are reported as mean +/− SEM. A p value of < 0.05 was considered significant, and all are two-tailed.The anti-proliferative effects of chemotherapeutic agents on CAFs were determined by a 3--2,5-diphenyltetrazolium bromide (MTT) assay, as previously described . Multiplex bead assays were done with BioSource Multiplex Assays for Luminex (Invitrogen) in a 96-well format according to the BioSource protocol. CAF concentrations were calculated based on a standard curve derived by performing six serial dilutions of a protein standard in assay diluent. Samples were tested in duplicate, and the mean value was used for analysis. If the mean of the duplicate values for all factors in an individual sample varied by >25%, the sample was retested. Individual factors were excluded from analysis if ≥50% of the samples were out of range or extrapolated. When out-of-range values were included in the analysis, the highest value (measured or extrapolated) across all samples was substituted for values above the upper limit of detection. For values below the lower limit of detection, half the lowest value was substituted.TACs were measured by multiplex bead assay (36 factors) as previously described . Reactivity to TrkB or BDNF was assessed immunohistochemically based on the percentage of positive cells in 10 consecutive high-power fields (1000 cells) and the intensity of staining graded from 0 to 3+ . Positive  and negative  controls were run in parallel for all experiments. The results were analyzed with Fisher’s exact test to determine the statistical significance of staining and tumor differentiation across experimental groups. A p value of <0.05 was considered significant. TUNEL (Roche), PCNA, Ki67  and E-cadherin staining and analysis were performed as previously described , 33.Fresh-frozen and paraffin-embedded HNSCC tumors were obtained from the MD Anderson Cancer Center Head and Neck Tumor Tissue Repository under an Institutional Review Board approved protocol. The HNSCC tissue array was designed as previously described 4 cells were plated in cell culture insert wells  or Matrigel-coated wells (BD Biocoat) under the described conditions. After 24 or 48 hours, the unmigrated cells on the upper chamber were removed and inserts were fixed and stained with Diff-Quik. For co-culture experiments, CAF cells were seeded into the lower chamber and tumor cells into the upper chamber of cell culture inserts, as above. After 24 or 48 hours, the unmigrated cells on the upper chamber were removed and inserts were fixed and stained with Diff-Quik. Images were captured  and the degree of migration or invasion was determined by the average number of migrated cells in five 100× fields . Experiments were performed in triplicate and repeated three times. Measurements were taken at indicated time-points and were quantified with ImagePro. Differences between groups were analyzed by utilizing either conventional Student’s t test or ANOVA followed by post hoc comparisons based upon modified Newman-Keuls-Student procedure, where appropriate. Results are reported as mean +/− SEM. A p value of < 0.05 was considered significant and all are two-tailed.Cells were serum-starved overnight and then 5×10. Cells (3×104) were seeded in each of the reservoirs, grown overnight, and wound closure measurements were taken 24 hours following removal of the inserts. Experiments were performed in triplicate and repeated three times. Measurements were taken at indicated time-points and were quantified with ImagePro. Differences between groups were analyzed by utilizing either conventional Student’s t test or ANOVA followed by post hoc comparisons based upon Bonferroni’s Multiple Comparison Test, where appropriate. Results are reported as mean +/− SEM. A p value of < 0.05 was considered significant, and all are two-tailed.Wound healing assay was performed using IBIDI  culture insert method in a six-well plate, as previously described . The next day, cells were washed twice with serum-free DMEM and cultured with serum-free medium containing BDNF (100ng/mL) for 24 Hrs. Supernatants were collected and centrifuged to remove cellular debris and the supernatant protein was concentrated with the Centricon 3 system.Cells were seeded in 10-cm dishes and grown to 70% confluency as described previously The PGL3 construct containing the MMP-9-luciferase reporter gene was kindly provided by Dr. Gary Clayman (MD Anderson Cancer Center). HNSCC cell lines were transfected with the pGL3-MMP9-Luc construct in six-well plates, as described above. Cells were treated with CAF-derived conditioned media from CAF cells subjected to various treatment conditions as explained elsewhere. Cell lysates were prepared from each of the treatments and the luciferase activity was measured using Promega dual luciferase assay kit as per the instructions of the manufacturer . All of the reporter assays were done in triplicate and repeated three times.3) were suspended in DMEM agar mixture  and plated over the bottom layer of the 0.6% agar-medium mixture. After 12 days, plates were stained with 0.005% crystal violet and colonies larger than 100uM were counted.Six-well culture plates were coated with 1 ml bottom agar mixture  and allowed to solidify at room temperature. Cells  (2.5×104), combination of OSC19-Luc and CAF, combination of OSC19-Luc and CAF with NT shRNA or as a combination of OSC19-Luc and CAF with BDNF shRNA in a ratio of 1:1 or 1:5 HNSCC:CAF.Male athymic nude mice (NCI-nu), aged 8–12 weeks, were purchased from the Animal Production Area of the National Cancer Institute-Frederick Cancer Research and Development Center . The mice were used in accordance with Animal Care and Use Guidelines of The University of Texas M. D. Anderson Cancer Center under a protocol approved by the Institutional Animal Care and Use Committee. For establishment of HNSCC tumors, cells were injected into the tongue of athymic nude mice with 2.5×10p value of < 0.05 was considered significant. Survival was calculated by the method of Kaplan and Meier. For the experimental model of metastasis, 1×105 cells were injected into the tail vein of mice. At the end of all experiments, mice were sacrificed and tissues were collected for H&E staining and immunohistochemistry (IHC) analysis. TUNEL and Ki67 IHC staining and analysis were performed as previously described .Tumor volume was measured weekly, and the differences between tumor volumes were evaluated by the non-parametric Mann–Whitney test. Results are reported as mean +/− SEM. A Xenogen, Alameda, CA) after intraperitoneal administration of D-luciferin . Results from the in vivo Luciferase assays were evaluated by the non-parametric Mann-Whitney test. Results are reported as mean +/− SEM. A p value of < 0.05 was considered significant and are two-tailed.Mice were imaged weekly with the IVIS Imaging System . Light microscopic analysis of three CAF cell lines  revealed cellular morphologies that were consistent with mesenchymal stromal cells . The molin vitro aggressive behavior of HNSCC.To determine whether the CAF-tumor synapse had unilateral functionality, we studied the impact of CAFs on the behavior of carcinoma cells in various cell-based assays. We first observed that secreted mediators from CAFs induced robust growth of HNSCC tumor cells in culture, when compared to standard concentrations of growth media . We nextin vivo impact of CAF pathophysiology in the tumor microenvironment, we next extended these observations to animal models of lymphatic metastasis. Mice were injected with tumor cells alone, or were co-injected with CAF cells at various concentrations, utilizing the flank and orthotopic tongue models. In the flank model, the incidence of metastasis to regional nodes was enhanced when tumor cells were co-injected with CAFs. Utilizing either a dilution of 1:1 or 3:1, co-injection with CAF cells was associated with a statistically significant increased incidence of lymphatic metastasis  or siRNA targeting BDNF (siRNA-BDNF) was added to HNSCC cells growing in culture. The wound scratch assay revealed significant impairment of HNSCC migration under conditions of BDNF knockdown in the CAF-CM, suggesting that potentiation of cellular motility is due in part to paracrine communication between tumor and stromal cells through BDNF-TrkB . We nextin vitro conditions. We first stably inhibited the expression of BDNF in the CAF compartment under in vitro conditions with sh-RNA constructs. Cancer cells were exposed to medium from either BDNF-depleted CAFs (BDNF-shRNA) or non-targeted CAFs (NT-shRNA). In Matrigel invasion experiments, tumor cells exposed to BDNF-depleted CAF media has significantly decreased cellular invasion compared to media from non-targeted CAFs. This effect, however, was reversed when exogenous recombinant BDNF was added to the experimental conditions  To test the hypothesis that MMP-9 expression in the tumor compartment can be modulated by BDNF sourced from the CAF compartment, we explored the impact of alterations in the fibroblast secretome on this pathway. After transfection with an MMP-9 promoter construct linked to a luciferase reporter gene, HNSCC cell lines were exposed to conditioned media from either NF or CAF cells. When analyzed by luciferase activity, HNSCC cells exposed to CAF-CM had substantial upregulation of MMP-9 promoter activity compared to NF-exposed cell lines  or a non-targeting sequence (shRNA-NT), CAF cells were co-injected at a 5:1 ratio with HNSCC cell lines in the orthotopic tongue model. Co-injection of CAFs harboring an shRNA-NT construct with HNSCC cells demonstrated enhanced tumor growth, compared to co-injection of CAFs harboring an shRNA-BDNF construct. The suppression of BDNF in the CAF compartment led to decreased tumor growth, suggesting that CAF-derived BDNF is a critical mediator of in vivo tumor growth. These findings also support the notion that BDNF is required for tumor growth, but is not a mediator of tumor engraftment.+1 score) in 17% of tumors that were analyzed. On univariate analysis, the development of metastasis was significantly associated with BDNF expression, although an effect on survival was not demonstrable. We further identified that expression of LYVE-1 was significantly associated with BDNF expression in human HNSCC, suggesting that BDNF upregulation may induce LYVE-1-mediated lymphatic network establishment in this disease  phenotypic behavioral changes induced by CAFs are coordinated by BDNF-mediated TrkB signaling axis cascade that incites a pro-invasive expression profile (3) regional metastasis in mouse models of SCC is regulated in part by BDNF-TrkB signaling in the tumor microenvironment via CAFs (4) stromal-specific BDNF expression predicts outcomes for patients with SCC.In this study, we identify novel mechanisms for fibroblast-induced metastasis in human cancers, primarily via a paracrine neurotrophin axis in the tumor microenvironment. These conclusions are based upon the following critical findings: (1) human-derived cancer associated fibroblasts potentiate , 27. We have also demonstrated the contribution of BDNF and TrkB to a chemo-resistant phenotype , which is associated with the potentiation of regional and distant metastases , 40. More recently, a provocative study of glioblastoma demonstrated that Trk receptors are targets for stromal-derived NTs the microenvironment . However, what has been lacking is an understanding of how CAFs contribute to the development of metastasis to the regional lymph nodes. In this study, we sought to directly link neurotrophin secretion from CAFs to the development of lymphatic metastasis.Neurotrophin and their receptors have been directly implicated in tumor progression and hematogenous metastasis. We previously demonstrated that TrkB activation induced EMT and invasion in animal models of OCSCC, and other studies indicate that TrkB overexpression directly mediates the metastatic phenotype in murine tail-vein and subcutaneous models of cancer progression –46. Other work has revealed a relationship between CAFs and lymphatic metastasis, but whether this remains a biological association is unclear, and a precise linkage mechanism remains elusive. Circumstantial evidence for this includes: (1) VEGF-C expression in CAFs is associated with increased nodal metastasis , (2) inhibition of CAF-secreted ECM components is associated with abrogated lymphangiogenesis  and (3) direct molecular targeting of CAFs inhibits lymphangiogenesis though an immunomodulatory mechanism. While these studies demonstrate the implicit importance of CAFs, the complex nature of human-tumor derived CAFs and their impact in murine tumor models remains poorly studied. Our data suggest that CAFs play a complex role in tumor progression that can be modulated by manipulation of the BDNF intercellular signaling system, mediated in part through alterations in pro-lymphangiogenic cascade.With regional lymph node metastasis often being the most important prognostic marker for patients with epithelial cancers, several studies have implicated VEGF-C as a biomarker of adverse prognosis for multiple cancers . Moreover, validation in epithelial cancers and in pre-clinical models has not been established. The mechanisms by which CAFs mediate growth in vivo have also been studied in some models, and a link to angiogenesis has been proposed. While MMP2- and MMP9-deificent embryonic fibroblasts co-injected with SCCA cells demonstrated suppressed tumor growth in a mouse model of cancer, an enhancement of tumor establishment was noted, suggesting that CAF are sufficient, but not necessary, for tumor development . This finding has been confirmed in other systems, including pancreatic  and lung cancers. In other animal models, fibroblast-derived MMP-1 activates the PAR-1 receptor, resulting in tumor invasion and angiogenesis in breast cancer . However, a link between CAFs and lymphatic metastasis has not been established to date, although substantial evidence supports a role for CAFs in vascular angiogenesis. The goals of this study were to determine whether the biological impact of CAFs extended to lymphatic metastasis in relevant models of human cancer. In this realm, out findings support the novel notion that the multi-step metastatic process in HNSCC is directly regulated in part by a unique CAF-tumor interface.There is evidence to suggest the presence of a BDNF signaling axis in the microenvironment of non-epithelial tumors, but the source of this ligand has been elusive . In a model of experimental metastasis, “priming” of tumor cells with fibroblast media potentiated engraftment of hematogenous metastases to lungs, a process mediated in part by TGF-β . In breast cancer, CAFs mediate pro- “estrogenic” effects through activation of the ERK pathway, suggesting a bi-directional interaction, although how this exists in mouse models has yet to be defined . Some of the most convincing in vivo data on a specific role for CAFs has been demonstrated in a FAP-deficient model of lung and colon cancer, with the demonstration that CAF-derived FAP directly modulates tumor growth and angiogenesis . One of the primary drivers of CAF-directed tumor progression appears to be an FGF-PGDFR intercellular circuit, which has been effectively targeted in a transgenic mouse model of cervical SCCA . Extending these findings to models of cutaneous SCCA, Erez et al identified that the step-wise progression from hyperplasia to cancer was controlled, in part, by an NF-κB inflammatory expression profile. Whether there is co-migration of CAFS with tumor cells to sites of metastasis remains unstudied, but an analysis of activated fibroblasts in rheumatoid arthritis revealed that pathologic fibroblasts are capable of “metastasis” to other joints, and similar phenotype in CAFs would not be impossible . In our study, we did not observe co-migration of CAFs to metastatic foci, and thus our findings suggest that the pro-metastatic impact of CAF occurs in the tumor micro-environment.In the CAF compartment, pre-clinical transgenic models have demonstrated that FAP regulates collagenase activity, and is important for angiogenesis and tumor growth in vivo have been studied in some models, and a link to angiogenesis has been proposed . Mediators of CAF-mediated metastasis in animal models of non-squamous cancers have been observed, and include TGF-β , estrogen-ERK , and FAP . In selected squamous cancers, FGF-PGDFR intercellular circuitry  and an NF-κB inflammatory expression profile  can control the step-wise progression from hyperplasia to cancer. While MMP-deficient embryonic fibroblasts co-injected with HNSCC cells demonstrated suppressed tumor growth in a mouse model of cancer, an enhancement of tumor establishment was noted, suggesting that CAF are sufficient, but not necessary, for tumor development . This finding has been confirmed in other systems, including pancreatic , lung and breast cancers . In our study, we have explored the novel hypothesis that lymphatic metastasis may be mediated through a BDNF-TrkB intercellular signaling system. It is likely that parallel interactions and signaling redundancies exist, and thus further exploration of how the neurotrophin signaling is modulated by FGF and FAP warrant further investigation.The mechanisms by which CAFs mediate tumor progression


Background: The modulation of arachidonic acid (AA) metabolism pathway is identified in metabolic alterations after hypoxia exposure, but its biological function is controversial. We aimed at integrating plasma metabolomic and transcriptomic approaches to systematically explore the roles of the AA metabolism pathway in response to acute hypoxia using an acute mountain sickness (AMS) model.Methods: Blood samples were obtained from 53 enrolled subjects before and after exposure to high altitude. Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry and RNA sequencing were separately performed for metabolomic and transcriptomic profiling, respectively. Influential modules comprising essential metabolites and genes were identified by weighted gene co-expression network analysis (WGCNA) after integrating metabolic information with phenotypic and transcriptomic datasets, respectively.Results: Enrolled subjects exhibited diverse response manners to hypoxia. Combined with obviously altered heart rate, oxygen saturation, hemoglobin, and Lake Louise Score (LLS), metabolomic profiling detected that 36 metabolites were highly related to clinical features in hypoxia responses, out of which 27 were upregulated and nine were downregulated, and could be mapped to AA metabolism pathway significantly. Integrated analysis of metabolomic and transcriptomic data revealed that these dominant molecules showed remarkable association with genes in gas transport incapacitation and disorders of hemoglobin metabolism pathways, such as ALAS2, HEMGN. After detailed description of AA metabolism pathway, we found that the molecules of 15-d-PGJ2, PGA2, PGE2, 12-O-3-OH-LTB4, LTD4, LTE4 were significantly up-regulated after hypoxia stimuli, and increased in those with poor response manner to hypoxia particularly. Further analysis in another cohort showed that genes in AA metabolism pathway such as PTGES, PTGS1, GGT1, TBAS1 et al. were excessively elevated in subjects in maladaptation to hypoxia.Conclusion: This is the first study to construct the map of AA metabolism pathway in response to hypoxia and reveal the crosstalk between phenotypic variation under hypoxia and the AA metabolism pathway. These findings may improve our understanding of the advanced pathophysiological mechanisms in acute hypoxic diseases and provide new insights into critical roles of the AA metabolism pathway in the development and prevention of these diseases. Hypoxia is a common phenomenon associated with multiple pathophysiological processes, including inflammation, necrosis, and apoptosis, and observed in various clinic conditions such as myocardial ischemia and reperfusion as well as stroke  , Lake Louise score (LLS), systolic blood pressure (SBP), diastolic blood pressure (DBP), and hemoglobin (HB) before and after high altitude exposure . These subjects were covered in our previous study , oxygen saturation  was used to preprocess the raw data, including peak detection, peak matching, matched filtration, and nonlinear alignment of data, with the default parameters .Metabolomics analysis was conducted on an Agilent 1290 Infinity LC system . Chromatographic separations were performed on an ACQUITY UHPLC HSS T3 C18 column  at 45°C. The metabolomics detection was performed totally in accordance to the manufacturers' protocols of all devices. Raw LC-MS data were converted to mzData formats via Agilent MassHunter Qualitative software . The program XCMS (version 1.40.0) (https://www.r-project.org). The model of partial squares discriminant analysis (OPLS-DA) was carried out to separate pre- and post-hypoxia in metabolic profiling. Variable importance project (VIP) was used to reveal discriminatory metabolites which were enrolled in the classification effects of hypoxia was calculated. In addition, adjusted p-value from the paired t-test and fold-change value (FC) were executed to discovery dominant metabolites. Pathway analysis was established by MetaboAnalyst platform .The resulting matrix from 53 subjects comprising retention time, mass-to-charge ratio (m/z), and normalized ion intensities was introduced into the subsequent analysis based on R platform . The data in this study were presented as the mean ± SD. A two-tailed p value < 0.05 was considered as significant, unless specifically indicated.Differences before and after hypoxia exposure were determined by paired 2, reflective of the oxygen concentration in the blood, significantly declined under hypoxia environment , indicative of the comprehensive response manner to acute hypoxia stimuli. A significant increase in LLS was observed for all individuals .The response of subjects to acute hypobaric hypoxia was assessed by various physiological parameters. As shown in Figure 2, LLS, and HB. To further explore influential metabolites in these modules, a volcano plot for these molecules was obtained . To further clarify the mechanisms underlying the AA metabolism pathway after hypoxia exposure, genes with the highest connectivity from the co-expression network and a correlation coefficient >0.65 to at least one molecule among 13 metabolites in the AA metabolism pathway are circled in red in Figure The physiological characteristics corresponding to SpOA map of metabolites in AA metabolism pathway was constructed in Figure p < 0.05, Figure GGT1, PTGES, PTGS1, CBR1, and ALOX12 encoding key enzymes of the AA metabolism pathway. The effects of these genes on AA metabolism pathways are labeled with a yellow triangle in Figure We validated our findings in another population of GSE52209 from GEO database that comprised 14 subjects as the control group and 17 individuals suffering from mal-adaptation to high altitude. In comparison to individuals that adapted to hypoxia, those with poor response to acute hypoxia showed a significant upregulation , and cysteinyl leukotrienes (Cys–LTs) in AA metabolism pathway after hypoxia exposure. These molecules were important sources of pro-inflammatory mediators. The elevation in the inflammatory response and oxidative stress may be important in AMS pathogenesis (Boos et al., The increase in metabolites indicates the upregulation in sub-pathways activated by COX, lipoxygenase, and PLA2—the key enzymes of the AA metabolism pathway (Wong et al., 2. These observations suggest impaired cardiopulmonary and hemoglobin function (Fuehrer and Huecker, dp/dt (Oshima et al., In our study, we demonstrated a crosstalk between response manners to acute hypoxia and expression level of the AA metabolism pathway. The excessive increase in the AA metabolism pathway after hypoxia stimuli may reflect mal-adaptation to hypoxia stimuli, as evident from the elevated levels of HR, HB, and LLS as well as the decreased level of SpOThe protective effects of anti-inflammatory molecules are increasingly recognized for the prevention of AMS. Apigenin targeting IL-1β, IL-6, and TNF-α was demonstrated to be effective in reducing the damage caused by acute hypoxia (Du et al., In summary, our integrated analysis of metabolomic and transcriptomic profiling revealed that the AA metabolism pathway was one of the most pivotal alterations after acute hypoxia exposure and may account for variations in response patterns to hypoxia stimuli. The detailed description of the AA metabolism pathway may offer the basis for follow-up mechanisms and drug screening.GSE103940.All data have been submitted to GEO under the accession YG and JC: Conceived and designed the study; CL and BL: Oversaw laboratory analyses and contributed the statistical analysis; LL, E-LZ, and BS: Contributed to sample and physical data collections; CL: Drafted the report. All authors reviewed and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The epidemiological and programmatic implications of inclusivity of HIV-positive males in voluntary medical male circumcision (VMMC) programs are uncertain. We modeled these implications using Zambia as an illustrative example.We used the Age-Structured Mathematical (ASM) model to evaluate, over an intermediate horizon (2010–2025), the effectiveness (number of VMMCs needed to avert one HIV infection) of VMMC scale-up scenarios with varying proportions of HIV-positive males. The model was calibrated by fitting to HIV prevalence time trend data from 1990 to 2014. We assumed that inclusivity of HIV positive males may benefit VMMC programs by increasing VMMC uptake among higher risk males, or by circumcision reducing HIV male-to-female transmission risk. All analyses were generated assuming no further antiretroviral therapy (ART) scale-up.The number of VMMCs needed to avert one HIV infection was projected to increase from 12.2 VMMCs per HIV infection averted, in a program that circumcises only HIV-negative males, to 14.0, in a program that includes HIV-positive males. The proportion of HIV-positive males was based on their representation in the population . However, if a program that only reaches out to HIV-negative males is associated with 20% lower uptake among higher-risk males, the effectiveness would be 13.2 VMMCs per infection averted. If improved inclusivity of HIV-positive males is associated with 20% higher uptake among higher-risk males, the effectiveness would be 12.4. As the assumed VMMC efficacy against male-to-female HIV transmission was increased from 0% to 20% and 46%, the effectiveness of circumcising regardless of HIV status improved from 14.0 to 11.5 and 9.1, respectively. The reduction in the HIV incidence rate among females increased accordingly, from 24.7% to 34.8% and 50.4%, respectively.Improving inclusivity of males in VMMC programs regardless of HIV status increases VMMC effectiveness, if there is moderate increase in VMMC uptake among higher-risk males and/or if there is moderate efficacy for VMMC against male-to-female transmission. In these circumstances, VMMC programs can reduce the HIV incidence rate in males by nearly as much as expected by some ART programs, and additionally, females can benefit from the intervention nearly as much as males. Randomized controlled trials (RCT) reported approximately 60% efficacy for male circumcision against heterosexual HIV acquisition among males –3. CurreWHO and UNAIDS recommend that HIV testing and counseling (HTC) be offered to all clients presenting for VMMC, as part of the comprehensive package of HIV prevention services . While Vmale-to-female HIV transmission). The evidence is mixed on this efficacy . Am. Am38]. Scale-up of other interventions such as ART or condom distribution may potentially diminish the benefits of VMMC scale-up programs. However, our earlier modeling study showed that even with an optimistic ART scale-up scenario, VMMC in an HIV hyper-endemic setting such as Zambia will remain an impactful and effective intervention. Also, scale-up of ART is not likely to change our predictions of the programmatic implications of including HIV-positive males in VMMC programs, as our study focused on the relative differences in VMMC effectiveness across scenarios rather than the absolute changes in VMMC effectiveness.relative difference in VMMC effectiveness [In our model we did not explore other relevant components of VMMC efficiency such as that of reduction in HIV testing costs that may shift the trade-offs in balance of a broad recruitment strategy. Also, the implications of inclusion of HIV-positive males in VMMC programs was only assessed at a national level rather than at subregional and/or rural versus urban levels. Nonetheless, an earlier modeling study indicated that geographical heterogeneities do not have a considerable effect on the tiveness .Furthermore, though we do not yet know the exact magnitude of the efficacy of male circumcision against male-to-female HIV transmission, a mounting body of evidence suggests that the effect is sizable , 20, 65.Improving the inclusivity of all males in VMMC programs is likely to enhance the programs’ effectiveness. Enhanced effectiveness would occur with moderate increases in the uptake of VMMC among males at higher risk and/or if male circumcision has moderate efficacy against male-to-female HIV transmission. In these circumstances, the one-time VMMC intervention can reduce the HIV incidence rate within a population by as much as some lifelong ART programs do. The reduction in incidence rate among females under these conditions can also be nearly as high as that among males.In light of these results, VMMC programs may wish to attract males regardless of their willingness to test for HIV and learn their HIV status. Program planners should not hesitate to recruit VMMC clients among populations at higher risk for HIV infection for fear of inadvertently increasing participation by HIV-positive males. Indeed, we believe that restricting recruitment in this way could affect the uptake of VMMC among both HIV-positive and -negative males, which would undermine a program’s overall impact. We believe that recruiting clients regardless of their perceived HIV status or willingness to be tested could reduce stigma and increase uptake of VMMC among higher-risk males, leading to more effective VMMC programs. Better understanding of the factors that affect the uptake of VMMC among males will allow programs to overcome the plausible lower uptake among higher-risk males while maintaining their links to other HIV care and treatment services, such as HTC.S1 Text(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.S4 Fig(DOCX)Click here for additional data file.


Circulating microRNAs in the blood may provide diagnostic and prognostic information about canine neoplastic diseases, and their profiles may be conserved between human and canine species. We performed RT-qPCR to obtain the profiles of circulating plasma microRNA-214 and -126 in total 181 cases of canine neoplastic diseases and healthy controls. MicroRNA-214 levels were high in 2 epithelial tumours (thyroid and mammary carcinomas) and 4 non-epithelial tumours . In contrast, microRNA-126 levels were high in 6 epithelial tumours  and 4 non-epithelial tumours . The diagnostic potential of microRNA-214 was relatively high in sarcomas, whereas that of microR-126 was high in most types of the tumours. MicroRNA-214 and -126 were prognostic predictors in 2 groups  and 3 groups , respectively. Additionally, the microRNA levels did not show a strong correlation with the other clinical parameters. In conclusion, circulating microRNA-214 and -126 have the potential to be diagnostic and prognostic biomarkers for canine neoplastic diseases. Furthermore, their profiles may be key references as well for exploring novel biomarkers for human cancers. However, clinically available low-invasive biomarkers for canine tumours have yet to be identified. To date, some blood proteins and enzymes, such as canine C-reactive protein (CRP) and thymidine kinase, have been studied as low-invasive biomarkers for canine tumours3; however, these protein-based markers were found to be difficult for fully meeting the clinical demand as tumour biomarkers because their activities are easily affected by a change in the storage temperature and numerous other clinical conditions including inflammation. Identifying novel low-invasive biomarkers for canine tumours is thus of great interest in veterinary clinical oncology.Low-invasive biomarkers for obtaining diagnostic and prognostic information may have considerable importance in determining the treatment strategy for patients with neoplastic disease. Several blood-based biomarkers, such as carcinoembryonic antigen (CEA)4. Canine spontaneous tumours have an additional unique feature of maintaining the tumour microenvironment, including tumour heterogeneity, intact immune system, abnormal vasculature, and intratumoral hypoxic gradient, whereas conventional mouse xenograft models lack these features5. Furthermore, the domestic dog frequently develops several tumour types that are extremely rare in humans; for instance, the incidence of sarcomas, which are malignancies derived from mesenchymal cells, is 4 times higher in dogs  than in humans . Given the similarities and unique features, canine spontaneous tumours have the potential to be useful models in biomarker research; and thus, developing biomarkers for canine neoplastic diseases might help toward the discovery of novel biomarkers for human neoplastic diseases.Identifying biomarkers for canine neoplastic diseases is important with respect to human oncology as well. Human and canine spontaneous tumours share many essential biological and clinical features, such as their genetics, histopathological morphology, and malignant behaviours to invade and metastasise to other organs8. Cancer cells not only show dysregulation of miRNAs but also release these miRNAs into the bloodstream9. Such “circulating miRNAs” are stabilised by their binding proteins10 or extracellular vesicles (EVs), such as exosomes and microvesicles (MVs)11; and thus, they are resistant to degradation by RNase. The levels of circulating miRNAs can accurately reflect the number of tumour cells, response to treatment, clinical stages, and tumour grades12. In addition, the sequences of mature miRNAs are broadly conserved among diverse species13. Therefore, circulating miRNAs have a great potential to be common diagnostic and prognostic biomarkers for both human and canine neoplastic diseases.MicroRNAs (miRNAs), a class of short-noncoding RNAs, play indispensable roles in tumorigenesis by controlling the differentiation, proliferation, and cell death of cancer cells16; however, the miRNAs suitable as biomarkers to evaluate various human and canine tumours are limited to those being released into the blood and regulating fundamental mechanisms of tumour pathogenesis in both species. MicroRNA-214 (miR-214) and microRNA-126 (miR-126) regulate angiogenesis, proliferation, migration, and cell death of cancer cells; and thus, dysregulation of these 2 miRNAs critically influences tumour progression18. Furthermore, circulating miR-214 or -126 levels are increased in 6 human tumours  and a canine sarcoma (canine hemangiosarcoma25). Considering the broad influence of miRNAs on cancer pathogenesis beyond histologic types and species, we hypothesized that circulating miR-214 and -126 could be potential low-invasive biomarkers common for diverse human and canine tumours.A large number of miRNAs are dysregulated in various human and canine tumours18; and few studies have attempted to obtain the miRNA profiles in a broad spectrum of tumour types. This is because humans develop limited types of cancers, and as such, it may be difficult to collect a wide variety of cancers, especially the rare ones such as sarcomas. In veterinary medicine, although dogs develop a broad range of tumours, levels of circulating miR-214 and -126 have not been explored except for our previous report on canine hemangiosarcoma25. We thus addressed this issue in our present study by evaluating the levels of circulating miR-214 and -126 in a wide variety of canine neoplastic diseases. The purpose of this study was to assess the potential of circulating miRNA-214 and -126 for use as diagnostic and prognostic biomarkers in various canine neoplastic diseases, as well as for obtaining referential information for exploring novel biomarkers in human cancers.In order to validate the versatility and practicability of miRNA-based biomarkers, it is necessary to assess the overall profiles between diverse tumour types in both humans and dogs. However, prior studies on human cancers have mainly focused on determining the levels of the miRNAs in a single or small population of specific tumours without a comparison between the tumour typeshttp://www.mirbase.org)26. The sequence homology of human and canine mature miRNAs was evaluated by using Microsoft Excel .Sequences of human and canine mature miRNAs were obtained from miRBase  according to the standard protocol. Three hundred µL frozen plasma samples were thawed and centrifuged for 3 min at 11,000 × g to remove residual cell debris. Blood proteins in the supernatant were precipitated by using a reagent in the kit and removed by centrifugation. After adjustment of the binding conditions with isopropanol, circulating RNAs were bound to an miRNA collection column. To avoid contamination by cell-free plasma DNAs, a recombinant DNase in the kit was applied to digest these DNAs on the column. The miRNA was then eluted into 10 µL of RNase-free water. The yield of circulating miRNA from plasma was normalised by using the same amount of plasma sample (300 µL) in each extraction. The purified miRNA was directly and immediately used for the following RT process to prevent degradation of RNA before PCR step. Common methods for RNA integrity and quality check before the RT process, such as a microvolume spectrophotometer and denaturing polyacrylamide gel electrophoresis, could not be employed in the present study due to the following reasons. The yield of total RNA from plasma is usually very small, and a microvolume spectrophotometer is hardly able to detect them, unlike cellular total RNA from tissues and cells, or serum having contamination of abundant platelet-derived cellular RNAs27. Denaturing polyacrylamide gel electrophoresis and other methods assessing 18S and 28S ribosomal RNA are also not available to evaluate the quality of circulating miRNA because total RNA from plasma does not have such predominantly abundant RNA. cDNA was amplified immediately after the RT reaction had been finished to minimise the degradation before the PCR reaction. The RNA quality was assessed by measuring circulating miR-16 levels. miR-16 is abundantly and stably found in plasma similar to ribosomal RNA in cellular RNAs, and thus, the levels should reflect the grade of RNA degradation and thus the quality of the plasma samples.Total circulating RNA was extracted from plasma samples by using the NucleoSpin® MicroRNA Assays  for miR-214 (AB Assay ID 002306), miR-126 (AB Assay ID 002228) and miR-16 (AB Assay ID 000391) with the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems®). miR-214, -126, and -16 were individually reverse-transcribed to cDNA by using the looped-RT primers that specifically reverse-transcribes each miRNA and add extensional sequences for the following PCR amplification. Each cDNA replicate was made in single. The RT condition was as follows. Step 1: 4 °C for 3 min; Step 2: 16 °C for 30 min; Step 3: 42 °C for 30 min; Step 4: 85 °C for 5 min followed by a 4 °C hold. The cDNA unique to each miRNA was subsequently amplified by using pre-designed forward and reverse PCR primers and TaqMan minor-groove-binder probes. These primers specifically bind to cDNA containing the sequences of each miRNA with the extension by the looped-RT primer. cDNA was diluted to 10 times at the final concentration with THUNDERBIRD® Probe qPCR Mix  and DNase/RNase-free distilled water. The cDNA was amplified with the measurement of fluorescence using a TaKaRa Thermal Cycler Dice® Real Time System I . The cycle conditions used in the PCR step was as follows. Step 1: 95 °C for 30 sec; Step 2: 95 °C for 5 sec; Step 3: 60 °C for 1 min; Repeat Steps 2–3 for 50 cycles. The PCR replicate was made in triplicate. The amplification efficiency of the qPCR reaction was examined in several runs and confirmed to be high enough (96–100%), whereas the efficiency was not evaluated in all runs or samples.An miRNA reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed for measuring levels of circulating miR-214, -126, and -16. For the accurate detection and quantification of these short miRNAs having only 22 nucleotides, we performed a looped-primer RT-qPCR using the TaqMan28. The Ct values were identified at the cycle of maximum fluorescence acceleration, the beginning point of the log-linear phase in the amplification curve.The threshold cycle (Ct) was automatically calculated by the second derivative maximum method to minimise the errors due to variation of a manual threshold determination and differences of background fluorescence in the samples and runs25. We considered that miR-16 was a reasonable internal control for our purposes due to the following reasons. miR-16 is conserved between human and canine , platelet (Plat.), fibrinogen (Fibn.), prothrombin time (PT), activated partial thromboplastin time (APTT), blood urea nitrogen (BUN), creatinine (Cre.), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and CRP, was collected at the first visit as well as at the time of plasma sample collection. Detailed information regarding histopathological diagnosis and breeds are given in Supplemental Table 31 , which assesses the optimal cut-point based on Chi-square values defined by the log-rank test. The X-tile software used in this study was version 3.6.1.Survival information including survival time and cause of death was collected by a follow-up survey. Cases euthanized or in which death occurred by accident were excluded from the survival analysis. The Kaplan-Meier method and the log-rank test were used to assess the correlations between the levels of circulating miRNAs and survival. GraphPad Prism 7  was used for the illustration of Kaplan-Meier survival curves and calculation of the P-values. The optimal cut-off values dividing the cases into the groups with high and low circulating miRNA levels were calculated by using X-tile plot softwarehttp://www.cytoscape.org). For further assessment of the correlation between 2 variables, a scatter plot was constructed and linear regression analysis was performed by using GraphPad Prism 7 . The statistical methods used in this study are summarised in Supplemental Table JMP Version 12, 64-bit  or GraphPad Prism 7  was used for each statistical analysis. For single comparisons, a nonparametric Mann-Whitney U test was used for comparing the medians of the 2 groups. For multiple comparison, the Kruskal-Wallis test was first used for examining whether the samples originated from the same distribution; then, Steel’s test was performed as the post-hoc test for comparing several groups with a control. The cluster analysis, dendrogram, and heatmap were illustrated by using JMP Version 12, 64-bit (SAS Institute Inc.). The clusters were determined by Ward’s method. For sets of categorical data, a chi-square test was used to examine whether the difference was significant. Receiver operating characteristic (ROC) curve analysis was used to calculate the area under the curve (AUC) value for assessing the validity of using circulating miRNAs to discriminate disease cases from healthy subjects. The Youden index was used for determining the optimal cut-off point to calculate the specificities and sensitivities in ROC curve analysis. Multivariate correlation analysis was performed to examine the correlation between miRNA levels and the other clinical parameters using JMP Version 12, 64-bit (SAS Institute Inc.). Calculated correlations were summarised to a correlation map by using Cytoscape  canine miRNAs showed a perfect match with human homologs , non-epithelial (n = 77), miscellaneous (n = 14) tumours, and control cases (n = 22) were evaluated Fig.  or weighWe next performed miRNA RT-qPCR to determine the profiles of circulating miR-214 and -126 in the tumour-bearing and control cases. We confirmed that miR-16, the internal control, was stably detected in these samples  based on hierarchical cluster analysis using both circulating miR-214 and -126 levels. The result showed that the groups of epithelial, non-epithelial, miscellaneous tumours, and controls showed significantly different profiles , especially in the sarcomas  and aggressive sarcomas , but relatively low in the epithelial tumours (AUC = 0.75) and the group including all types of tumours  indicating that the regression curve explained only 11% of total samples , coagulation , kidney function (BUN and Cre.), hepatic injury , and inflammation (CRP). The results showed that the levels of circulating miR-214 and -126 were not strongly correlated with most of the clinical parameters evaluated Fig. . Only plles Fig. .Figure 732  and pancreatic carcinoma33 , the combination of CEA, NSE, CYFRA21-1, and CA-125 for lung cancer34 , and AFP for hepatocellular carcinoma35 . In addition, circulating miR-214 and -126 were prognostic predictors in 2 groups  and 3 groups , respectively. These 2 miRNAs did not show a strong correlation with the other clinical parameters. Given that many human and canine miRNAs share identical sequences, the profiles that we revealed here provide foundational information for developing novel miRNA biomarkers for both human and canine tumours; the future addition of other circulating miRNAs to these miRNAs has the potential to improve diagnostic and prognostic accuracy.Here, we revealed the profiles of circulating miR-214 and -126 in dogs with tumours of 17 histopathological subtypes and demonstrated that both miRNAs showed the potential of being biomarkers for canine neoplastic diseases. The levels of circulating miR-214 were increased in non-epithelial tumours, especially sarcomas. In contrast, the levels of circulating miR-126 were increased in a wide variety of canine tumours. Furthermore, circulating miR-214 and -126 showed high diagnostic accuracies in sarcomas  and all types of canine tumours , respectively. These specificities and sensitivities were relatively high compared to several biomarkers established in human medicine, such as CEA for colorectal cancer22 and prognostic20 biomarker for various human cancers. In this study, we showed that levels of circulating miR-214 increased, and showed high diagnostic accuracies, in the dogs with non-epithelial tumours, especially sarcomas such as hemangiosarcoma, osteosarcoma, chondrosarcoma, and histiocytic sarcoma. The cases of epithelial tumours, such as mammary and thyroid carcinomas, also showed increased levels of circulating miR-214. However, other epithelial cancers showed relatively low levels of circulating miR-214. These results suggest that circulating miR-214 would be a potentially useful diagnostic biomarker for canine sarcomas, whilst it would not be suitable for use as a screening test or diagnostics for the other types of tumour. Survival analysis showed that the adenocarcinoma cases and cases of non-epithelial tumours (excluding osteosarcoma) with a high level of circulating miR-214 had a short survival time, suggesting that circulating miR-214 would have prognostic value for these canine tumours. This result is consistent with previous reports indicating that miR-214 contributes to tumorigenesis by regulating apoptosis36, proliferation37, and migration37, as well as having an impact on prognosis20 in diverse human cancers. Canine osteosarcoma showed outstandingly high levels of circulating miR-214 compared to the other non-epithelial tumours; moreover, the non-epithelial tumours showed a significant survival difference only when the osteosarcoma population was excluded, suggesting that osteosarcoma has distinct profiles of circulating miR-214 from the other non-epithelial tumours. It was reported that miR-214 regulates osteoblastic differentiation of mesenchymal stem cells38, and the level of circulating miR-214 is increased in a genetically engineered mouse model of osteosarcoma and in human osteosarcoma patients20. Accordingly, the high levels of circulating miR-214 in dogs with osteosarcoma may suggest a firm association between miR-214 and tumorigenesis in canine osteosarcomas. Adenocarcinoma cases with high levels of circulating miR-214 had a significantly shorter survival time, although only a few cases of them classified into the group with a high or moderate increase of circulating miR-214 in the cluster analysis. This result suggests that miR-214 estimates the prognosis but is not suitable for the diagnosis of canine adenocarcinoma. Given that exosomal miR-214 induces angiogenesis39, abundant extracellular miR-214 may contribute to the local invasion and metastasis through accelerating tumour angiogenesis in canine adenocarcinomas.Circulating miR-214 has been suggested to be a diagnostic24. In this study, the levels of circulating miR-126 in various canine tumours increased and showed high diagnostic accuracies, although chondrosarcoma and lymphoma showed relatively low levels of this miRNA. The results suggest that circulating miR-126 could be a diagnostic and screening biomarker for a broad range of canine tumours except for chondrosarcoma and lymphoma. The high levels of circulating miR-126 associated with the short survival time of the dogs with adenocarcinoma, other epithelial tumours or melanoma, suggest that the level of circulating miR-126 could serve to estimate the prognosis for dogs with these tumours. Given that miR-126 regulates apoptosis40, cell migration41, and proliferation42 in various human cancers, the results suggest that miR-126 might also contribute to tumorigenesis in these canine tumours.Circulating miR-126, as well as miR-214, has been suggested to be a diagnostic biomarker for human neoplastic diseaseCirculating miR-214 and -126 showed different profiles between the epithelial and non-epithelial tumours in this study: the level of circulating miR-214 increased mainly in the sarcomas; in contrast, circulating miR-126 increased in most types of the tumours examined. These results suggest the further application of circulating miR-214 and -126 to determination of the origin of primary tumours, although more detailed information on the profiles will be necessary for such clinical application.43. In the current study, we measured the total circulating plasma miRNAs, which included both EV-carried and soluble miRNAs, and demonstrated that they also had the potential to be accurate biomarkers. Moreover, multivariate correlation analysis showed that the levels of circulating miR-214 and -126 showed no correlation with most of the clinical parameters investigated in this study. These results suggest that the levels of circulating miR-214 and -126 were independent of common concurrent diseases associated with neoplasia, such as anaemia, inflammation, and hepatic and renal disorders; thus, they might be able to accurately assess the state of the tumour without noise from concurrent disease. Only the platelet count showed a statistically significant correlation with the levels of circulating miR-126 (P < 0.0001); however, the platelet counts had a relatively small impact on determining the miR-126 levels (R2 = 0.1111). The results reflected a previous finding that platelets contain miR-126 in their cytoplasm44, and suggest that miR-126 could leak out into the plasma from the platelets. Therefore, the counts and disorders of platelets possibly affected the levels of circulating miR-126, although their impact might be negligibly small. Taken together, the results suggest that circulating miR-214 and -126, which include both EV-carried and non-EV-carried forms, would be biomarkers suitable for evaluation of the tumour state with minimal effects from any concurrent disease.Soluble (Non-EV-carried) miRNAs, unlike exosomal or MV-carried miRNAs, were once suggested to be unsuitable as biomarkers, because of their potential fragility and the unspecified influence of concurrent disease. However, more recent papers have indicated that soluble miRNAs are stabilised by binding proteins such as AGO1 and AGO2, and thus can be potentially stable biomarkers as well5. In the present study, the canine profiles of miR-214 and -126 were consistent with several previous reports on human cancers. For instance, circulating miR-214 was increased in canine mammary carcinomas and osteosarcomas; and these results agree with previous reports showing that miR-214 levels are increased in patients with breast cancer21 and osteosarcoma20. Also, the level of circulating miR-126 was increased in canine melanomas, which corresponds with the report that circulating miR-126 is also increased in human metastatic sporadic melanoma45. Furthermore, there are also other reports indicating that circulating miR-214 is increased in human gastric cancers22, ovarian cancers23, and myeloma19, and that circulating miR-126 also is increased in human non-small cell lung carcinoma24. However, we were not able to assess the similarities between these human tumour types and their canine homologs due to the small number of the latter included in this study (Supplemental Table 51. These findings lead us to conjecture that canine and human tumours share not only their clinical and histologic features but also the profiles of circulating miRNAs, although further validation is necessary. The canine profiles of neoplastic disease thus might serve as useful references for finding biomarkers in human tumours including sarcomas.Canine tumours have been advocated as an ideal model for human tumours in that they have biological and clinical behaviours, as well as a tumour microenvironment, similar to those for human tumours52, as well as that circulating miR-126 is increased in moyamoya disease46, allergic rhinitis, and asthma50 but decreased in atherosclerosis47, atrial fibrillation, heart failure51, and type-2 diabetes mellitus49. Future studies interrogating these miRNA profiles in non-neoplastic diseases will provide for the practical usage of these circulating miRNAs as cancer biomarkers. Third, it is still unclear whether these miRNAs are released from the tumour cells themselves. Our previous study in canine hemangiosarcoma suggested that circulating miR-214 and -126 are released from the tumour cells themselves: hemangiosarcoma cell lines released miR-214 and -126 into the surrounding environment, and surgical resection of the primary tumour decreased levels of these circulating miRNAs in the dogs with hemangiosarcoma25. In this present study, however, we evaluated only the levels of circulating miRNAs, not the levels in the primary tumour or in cell lines of each tumour type. Thus, in the other tumour types except for hemangiosarcoma, these miRNAs may be released from certain components of the tumour tissue, such as tumour-associated endothelial cells, inflammatory cells or fibroblasts, or by some condition associated with the neoplastic disease. The miRNAs, however, still have diagnostic or prognostic potential as tumour biomarkers whether they are released from the tumour cells themselves or not. Addressing the direct aetiology of the increase in these miRNA levels would accelerate our understanding of these miRNAs and their clinical usage. Finally, although miR-214 and -126 levels normalised by miR-16 showed a significant utility in canine cancer diagnosis and prognosis, there might be better internal controls other than miR-16 for these circulating miRNAs. Currently, the information regarding internal controls for normalising canine plasma miRNAs is very limited; however, further validation regarding the internal control might improve the diagnostic and prognostic potential of these miRNAs.We acknowledge several limitations in this study. First, although we collected a total of 191 cases in this study, some subcategories included a relatively small number of cases; thus, the results might not represent the actual clinical utilities in such subcategories. However, our results provide and overall perspective of the circulating miR-214 and -126 profiles in canine neoplastic disease, and would help in an additional large-scale study of each tumour type to determine the true clinical usage of the miRNAs in veterinary clinical oncology. Second, this study included only healthy controls but not any non-neoplastic disease controls, such as inflammatory, infectious, and endocrine disease. Although we demonstrated that the other established clinical parameters did not show strong correlation with the levels of circulating miR-214 and -126 in this study, it is still possible that the levels of these circulating miRNAs might increase or decrease in other non-neoplastic diseases: several studies examining human diseases reported that the level of circulating miR-214 is decreased in coronary artery diseaseIn conclusion, we have shown that circulating miR-214 and -126 have the potential to be diagnostic and prognostic biomarkers for canine neoplastic disease. Their profiles may also prompt future exploration for novel biomarkers for human cancer.Supplemental materials