Toward the isolation of exosomes by flow cytometry

In the last two decades the Extracellular Vesicles (EVs) field has attracted a lot of attention from the scientific community, especially after the discovery that EVs can shuttle functional proteins and nucleic acids between cells. Some recent studies have shown an association between tumorigenesis and increased exosomes production. Exosomes and their influence has also been reported in the establishment of new metastatic niches. Besides that, the EV field remains confusing due to numerous and ambiguous definitions, specially caused by the huge heterogeneity of the vesicles, both in composition and function. Extracellular vesicles are divided into microvesicles which are originated from the plasma membrane and exosomes which have an endosomal origin. For now, it is technically challenging to obtain a pure exosome fraction, free from non-vesicular components, due to the fact the extracellular milieu is quite complex and can contain microvesicles or apoptotic bodies similar in size and structure to exosomes. The two most used methods, ultracentrifugation and commercial kits, don’t show a good efficiency when distinguishing the exosomes fraction specifically from the microvesicles fraction. Due to this sub-optimal efficiency demonstrated by these two methods, we have decided to use Flow Cytometry to see if we can achieve better exosome purification. We will use Fluorescence-activated cell sorting (FACS) to purify endogenous exosomes. This would be quite challenging especially due to the exosomes size and heterogeneity but on the other hand, if we have success with our approach, it would be possible to do downstream analysis in order to know their protein composition, functions and elaborate some more studies to try to find some “exosome-specific” marker. This would have a huge impact in the pharmaceutical industry, both for diagnosis and therapy.Durante as últimas duas décadas, a investigação desenvolvida sobre Vesículas extracelulares (VE), atraíu o bastante interesse por parte da comunidade científica, especialmente após ter sido descoberto que as VE podem transportar proteínas funcinais e ácidos nucleicos entre diferentes células. Estudos mais recentes mostraram uma relação entre tumorogenese e um aumento na produção de exosomas. Estes foram também associados ao estabelecimento de novas metástases. Apesar de todas estas descobertas, o domínio das VE continua significativamente confuso, nomeadamente devido às numerosas e ambíguas definições utilizadas, especialmente devido ao facto da imensa heterogeneidade entre as diversas VE, tanto a nível de composição como de função. Vesículas extracellulares estão divididas em microvesículas, que são originárias da membrana plasmática, e exosomas que têm uma oigem endossomal. No presente, é tecnicamente bastante complicado de obter uma fracção de pura exosomas que não apresente componentes não vesiculares, principalmente pelo facto do meio extracellular ser bastante complexo e poder conter microvesícula e corpos apoptóticos semelhantes em termos de tamanho e estrutura. Os dois métodos mais usados, a ultracentrifugação e kits comerciais, não apresentam uma boa eficiência na distinção de exosomas, especialmente das microvesículas. Devido a esta eficiência sub-óptima demonstrada por estes dois métodos, decídimos usar a separação celular por citometria de fluxo (FACS) para proceder ao isolamento de exosomas endógenos. Este objectivo será bastante desafiador especialmente pelo tamanho e heterogeneidade dos exosomas mas, por outro lado, se formos suficientemente bem sucedidos na nossa abordagem, será possível realizar análises posteriores, de modo a conhecer a sua composição proteica, funções e partir para novos estudos de modo a tentar identificar um marcador molecular específico para exosomas. Isto teria um impacto significativo na indústria farmacêutica, tanto a nível de diagnóstico como terapêutico

Global variation in postoperative mortality and complications after cancer surgery: a multicentre, prospective cohort study in 82 countries

© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 licenseBackground: 80% of individuals with cancer will require a surgical procedure, yet little comparative data exist on early outcomes in low-income and middle-income countries (LMICs). We compared postoperative outcomes in breast, colorectal, and gastric cancer surgery in hospitals worldwide, focusing on the effect of disease stage and complications on postoperative mortality. Methods: This was a multicentre, international prospective cohort study of consecutive adult patients undergoing surgery for primary breast, colorectal, or gastric cancer requiring a skin incision done under general or neuraxial anaesthesia. The primary outcome was death or major complication within 30 days of surgery. Multilevel logistic regression determined relationships within three-level nested models of patients within hospitals and countries. Hospital-level infrastructure effects were explored with three-way mediation analyses. This study was registered with ClinicalTrials.gov, NCT03471494. Findings: Between April 1, 2018, and Jan 31, 2019, we enrolled 15 958 patients from 428 hospitals in 82 countries (high income 9106 patients, 31 countries; upper-middle income 2721 patients, 23 countries; or lower-middle income 4131 patients, 28 countries). Patients in LMICs presented with more advanced disease compared with patients in high-income countries. 30-day mortality was higher for gastric cancer in low-income or lower-middle-income countries (adjusted odds ratio 3·72, 95% CI 1·70–8·16) and for colorectal cancer in low-income or lower-middle-income countries (4·59, 2·39–8·80) and upper-middle-income countries (2·06, 1·11–3·83). No difference in 30-day mortality was seen in breast cancer. The proportion of patients who died after a major complication was greatest in low-income or lower-middle-income countries (6·15, 3·26–11·59) and upper-middle-income countries (3·89, 2·08–7·29). Postoperative death after complications was partly explained by patient factors (60%) and partly by hospital or country (40%). The absence of consistently available postoperative care facilities was associated with seven to 10 more deaths per 100 major complications in LMICs. Cancer stage alone explained little of the early variation in mortality or postoperative complications. Interpretation: Higher levels of mortality after cancer surgery in LMICs was not fully explained by later presentation of disease. The capacity to rescue patients from surgical complications is a tangible opportunity for meaningful intervention. Early death after cancer surgery might be reduced by policies focusing on strengthening perioperative care systems to detect and intervene in common complications. Funding: National Institute for Health Research Global Health Research Unit

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

© 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licenseBackground: Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide. Methods: A multimethods analysis was performed as part of the GlobalSurg 3 study—a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital. Findings: Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3·85 [95% CI 2·58–5·75]; p<0·0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63·0% vs 82·7%; OR 0·35 [0·23–0·53]; p<0·0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer. Interpretation: Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised. Funding: National Institute for Health and Care Research