Dissecting functional and molecular signatures of anti-tumour myeloid cells in vivo

Tese de mestrado, Oncobiologia, Universidade de Lisboa, Faculdade de Medicina, 2019The tumour microenvironment is a heterogeneous ecosystem that can be densely populated by immune cells, both of lymphoid and myeloid origin. Usually, up to 50% of the immune infiltrate can be constituted by a multitude of myeloid cells that, accordingly to environmental cues, can either act as antitumoural or pro-tumoural effector cells. Plasticity is a widely accepted hallmark of myeloid cells and to date there has been a disproportionate attention on pro-tumour over anti-tumour myeloid cell functions. However, we believe that more efforts should be put on understanding how to enhance the protective activity of myeloid cells in cancer. In this thesis, to study myeloid cells as they perform anti-tumour effector functions in vivo, we took advantage of their inherent capacity to respond to maturing agents such as TLR ligands and co-stimulatory agonists. We found that in the orthotopic E0771 mammary tumour model, the injection of TLR3 ligand plus anti-CD40 mAb was able to induce tumour regression in a macrophage-dependent manner. These macrophages were stimulated to produce specific pro-inflammatory cytokines and enzymes. In addition, CD8+ T cells were activated, thus promoting a cytotoxic response leading to the complete eradication of tumours in TLR3/anti-CD40 treated mice. These results led us to question whether other distinct TLR ligands could induce complete remission of the tumour, potentially dependent on different anti-tumour myeloid cell lineages. Indeed, we found that TLR4 ligand in combination with anti-CD40 mAb induced tumour elimination in a neutrophildependent manner. Altogether, in this thesis we were successfully able to shape the tumour microenvironment towards boosting the anti-tumour potential of macrophages and neutrophils using different and non-overlapping treatments. In sum, our results lay the groundwork for further studies that will combine unbiased approaches (transcriptomics) with in situ assessment of the biology, functionality and differentiation program of anti-tumour macrophages and neutrophils. In the longer run, we hope such knowledge will help to design new strategies to manipulate myeloid cells in order to unleash their anti-tumour potential and thus contribute to more efficient cancer immunotherapies.Ao longo dos ultimos anos, o aumento do conhecimento acerca do sistema imunitario e da sua importancia na destruicao das celulas tumorais tem levado ao desenvolvimento de novas estrategias imunoterapeuticas. Estas estrategias representam uma nova era na area da oncologia e tem como principal objectivo estimular e potenciar uma resposta imune. Porem, apesar de todos os resultados clinicos notaveis, uma fracao significativa dos pacientes nao responde a imunoterapia. Esta resistencia e o resultado da capacidade das celulas tumorais manipularem o microambiente e promoverem imunossupressao local permitindo-lhes escapar ao reconhecimento e eliminacao por celulas imunes. O microambiente tumoral tem sido alvo de grande atencao por parte dos investigadores por estar directamente envolvido na progressao tumoral. Para alem das celulas neoplasicas, este e tambem constituido por diversas populacoes celulares, tais como as do estroma ou das linhagens linfoide e mieloide do sistema imunitario. Habitualmente, mais de 50% da infiltrado imune e composto por celulas mieloides que, de acordo com as informacoes que recebem do microambiente, podem estimular ou inibir a resposta imunitaria anti-tumoral. As celulas mieloides tem uma grande plasticidade e ate hoje tem havido uma atencao desproporcional entre as celulas que promovem o crescimento do tumor (pro-tumorais) relativamente as que tentam travar o seu crescimento (anti-tumorais). Contudo, acreditamos que deveriam ser feitos mais esforcos de modo a perceber de que forma poderiamos aumentar a actividade protectora destas celulas. Para isso, neste trabalho utilizou-se um modelo ortotopico de celulas tumorais mamarias de ratinho (E0771) com o objectivo de estudar a actividade anti-tumoral das celulas mieloides durante o desenvolvimento tumoral in vivo, tirando partido do facto de estas serem capazes de responder a varios sinais, mais especificamente a ligandos de receptores de Toll (TLR) e moleculas co-estimuladoras. Nesta tese foi possivel demonstrar que in vivo, a injeccao intra-tumoral dos ligandos de TLR2/1 (Pam3CSK4), 2/6 (Pam2CSK4), 3 (Poly I:C), 4 (LPS) e 9 (CpG) em combinacao com o agonista anti-CD40, levou a remissao total do tumor. Alem disso, in vitro estes ligandos de TLR nao tem um impacto directo na proliferacao das celulas tumorais, o que significa que o efeito destes tratamentos devera ocorrer atraves da manipulacao das celulas celulas que constituem o microambiente tumoral. O Poly I:C e uma molecula de RNA de cadeia dupla associada a infecoes virais e que consegue ser muito semelhante a activacao que e feita aquando do aparecimento de um tumor. In vitro foi ainda possivel observar que este imunoestimulador, em combinacao com o anticorpo agonista anti-CD40, e um dos que menos afecta a expressao de moleculas do complexo maior de imunohistocompatibilidade classe I e II bem como dos marcadores imunossupressores dos ligandos de PD-1 das celulas tumorais. Com isto, decidimos dissecar os mecanismos especificos de accao pelos quais o tratamento intra-tumoral com o Poly I:C em combinacao com o anticorpo agonista anti-CD40 levavam a eliminacao do tumor. Desta analise, concluiu-se o efeito era dependente dos macrofagos (CD11b+ F4/80+ Ly6C+/-), uma vez que apos a deplecao destas celulas o tratamento deixou de funcionar. Descobrimos ainda que o tratamento levou a um aumento da producao de citocinas proinflamatorias, tais como IL-1 e TNF- ou mesmo enzimas, como o iNOS pelos macrofagos. Paralelamente a este aumento, houve uma diminuicao da expressao do marcador imunossupressor PD-L1 nos ratinhos que tinham sido tratados em comparacao com os nao tratados. Os nossos resultados mostraram ainda que, apos a re-injeccao das celulas tumorais E0771 na glandula mamaria de ratinhos onde o tumor ja tinha sido eliminado, este nao voltava a crescer. Este resultado sugere assim a capacidade de este tratamento com o ligando do TLR3 em combinacao com o agonista anti- CD40 criar uma memoria imunologica capaz de reconhecer o tumor como estranho e elimina-lo. Sabendo que as celulas T CD8+ estao associadas a memoria imunologica e sao altamente citotoxicas, colocamos entao a possibilidade de estas terem tambem um papel na eliminacao do tumor uma vez que a producao de TNF- e IFN- por estas celulas era aumentada pelo tratamento. As celulas T CD8+ mostraram entao ser fundamentais para a eliminacao o tumor, sendo que a sua deplecao evitava a regressao tumoral. dependente das celulas T CD8+. Alem disso, as celulas T CD8+, cuja producao de TNF- e IFN- e aumentada pelo tratamento, mostraram ser fundamentais para a eliminacao do tumor, uma vez que a sua deplecao evitava a regressao tumoral. Atraves destas descobertas foi entao possivel construir um modelo dividido em dois passos (two-step model) onde o tratamento com o ligando de TLR3 em combinacao com o anticorpo agonista anti-CD40 leva a activacao dos macrofagos e mais tarde, de uma forma directa ou indirecta (via celulas dendriticas), as celulas T CD8+ sao tambem activadas e tornam-se capazes de gerar uma resposta citotoxica contra os tumores, levando a sua total erradicacao nos animais tratados. Posteriormente analisamos a accao de outros ligandos de TLR na capacidade de inducao de remissao total do tumor e de activacao de outras celulas com propriedades propriedades anti-tumorais da linhagem mieloide: os neutrofilos. Chegamos a conclusao de que, em animais onde era feita a deplecao destas celulas atraves do anticorpo anti-Gr1, a injeccao intra-tumoral do Pam3CSK4, Pam2CSK4 e CpG com o anticorpo agonista anti-CD40 continuava a gerar uma resposta anti-tumoral resultando na regressao do tumor. Porem, com a injeccao intra-tumoral do ligando de TLR4 em combinacao com o agonista anti- CD40, em ratinhos depletados para neurofilos mostrou-se ineficaz na inducao de regressao tumoral. Estes resultados levaram-nos a concluir que a injeccao do ligando TLR4 com o anticorpo agonista anti-CD40 nao so levava a regressao total do tumor, mas tambem actuava directamente nos neutrofilos, tornando-os antitumorais. No entanto, sao necessarios estudos mais detalhados para perceber de que forma e que este tratamento funciona tal como foi feito para os macrofagos atraves do tratamento com o Poly I:C em combinacao com o anticorpo anti-CD40. Neste projecto fomos entao capazes de alterar o microambiente tumoral e, em particular, de estimular a capacidade anti-tumoral dos macrofagos e dos neutrofilos atraves de diferentes tratamentos. Em suma, os resultados nesta tese sao promissores e levam-nos a ambicionar por mais estudos que sejam capazes de decifrar quais os programas de transcricao e diferenciacao responsaveis por tornar estas celulas imunes antitumorais. No futuro, acreditamos que estes resultados nos poderao ajudar a desenvolver novas estrategias para manipular e aumentar o poder anti-tumoral destas celulas mieloides, de modo a melhorar a eficacia da imunoterapia do cancro

SARS-CoV-2 introductions and early dynamics of the epidemic in Portugal

Genomic surveillance of SARS-CoV-2 in Portugal was rapidly implemented by the National Institute of Health in the early stages of the COVID-19 epidemic, in collaboration with more than 50 laboratories distributed nationwide. Methods By applying recent phylodynamic models that allow integration of individual-based travel history, we reconstructed and characterized the spatio-temporal dynamics of SARSCoV-2 introductions and early dissemination in Portugal. Results We detected at least 277 independent SARS-CoV-2 introductions, mostly from European countries (namely the United Kingdom, Spain, France, Italy, and Switzerland), which were consistent with the countries with the highest connectivity with Portugal. Although most introductions were estimated to have occurred during early March 2020, it is likely that SARS-CoV-2 was silently circulating in Portugal throughout February, before the first cases were confirmed. Conclusions Here we conclude that the earlier implementation of measures could have minimized the number of introductions and subsequent virus expansion in Portugal. This study lays the foundation for genomic epidemiology of SARS-CoV-2 in Portugal, and highlights the need for systematic and geographically-representative genomic surveillance.We gratefully acknowledge to Sara Hill and Nuno Faria (University of Oxford) and Joshua Quick and Nick Loman (University of Birmingham) for kindly providing us with the initial sets of Artic Network primers for NGS; Rafael Mamede (MRamirez team, IMM, Lisbon) for developing and sharing a bioinformatics script for sequence curation (https://github.com/rfm-targa/BioinfUtils); Philippe Lemey (KU Leuven) for providing guidance on the implementation of the phylodynamic models; Joshua L. Cherry (National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health) for providing guidance with the subsampling strategies; and all authors, originating and submitting laboratories who have contributed genome data on GISAID (https://www.gisaid.org/) on which part of this research is based. The opinions expressed in this article are those of the authors and do not reflect the view of the National Institutes of Health, the Department of Health and Human Services, or the United States government. This study is co-funded by Fundação para a Ciência e Tecnologia and Agência de Investigação Clínica e Inovação Biomédica (234_596874175) on behalf of the Research 4 COVID-19 call. Some infrastructural resources used in this study come from the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by Fundação para a Ciência e a Tecnologia (FCT).info:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear un derstanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5–7 vast areas of the tropics remain understudied.8–11 In the American tropics, Amazonia stands out as the world’s most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepre sented in biodiversity databases.13–15 To worsen this situation, human-induced modifications16,17 may elim inate pieces of the Amazon’s biodiversity puzzle before we can use them to understand how ecological com munities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple or ganism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region’s vulnerability to environmental change. 15%–18% of the most ne glected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lostinfo:eu-repo/semantics/publishedVersio

Pervasive gaps in Amazonian ecological research

Biodiversity loss is one of the main challenges of our time,1,2 and attempts to address it require a clear understanding of how ecological communities respond to environmental change across time and space.3,4 While the increasing availability of global databases on ecological communities has advanced our knowledge of biodiversity sensitivity to environmental changes,5,6,7 vast areas of the tropics remain understudied.8,9,10,11 In the American tropics, Amazonia stands out as the world's most diverse rainforest and the primary source of Neotropical biodiversity,12 but it remains among the least known forests in America and is often underrepresented in biodiversity databases.13,14,15 To worsen this situation, human-induced modifications16,17 may eliminate pieces of the Amazon's biodiversity puzzle before we can use them to understand how ecological communities are responding. To increase generalization and applicability of biodiversity knowledge,18,19 it is thus crucial to reduce biases in ecological research, particularly in regions projected to face the most pronounced environmental changes. We integrate ecological community metadata of 7,694 sampling sites for multiple organism groups in a machine learning model framework to map the research probability across the Brazilian Amazonia, while identifying the region's vulnerability to environmental change. 15%–18% of the most neglected areas in ecological research are expected to experience severe climate or land use changes by 2050. This means that unless we take immediate action, we will not be able to establish their current status, much less monitor how it is changing and what is being lost

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030