Metabolic characterization of sarcomas

Tese de mestrado, Ciências Biofarmacêuticas, 2022, Universidade de Lisboa, Faculdade de Farmácia.Sarcomas são um grupo heterogéneo de tumores malignos com origem na transformação de células mesenquimais nos tecidos conjuntivos. Estes representam menos de 1% dos cancros adultos e abrangem mais de 170 subtipos, dos quais aproximadamente 87% provêm de tecidos moles e 13% do osso. A carcinogénese depende de alterações metabólicas e apresenta características comuns a um vasto espetro de diferentes cancros, tal como evasão à apoptose, potencial replicativo ilimitado, reprogramação do metabolismo energético, entre outros. Dois substratos metabólicos comummente utilizados pelas células cancerígenas para satisfazer as exigências biossintéticas e bioenergéticas são a glucose e a glutamina. Uma forma de estudar as características metabólicas dos tumores, incluindo os sarcomas, abrange diversas técnicas que serão descritas nesta Tese. A resultante Metabolómica é uma ampla análise quantitativa e qualitativa do metaboloma e uma das vantagens desta abordagem reside no facto dos metabolitos estarem a jusante de genes, transcrição e proteínas e, portanto, oferecem a representação mais próxima do fenótipo. Neste projeto, tínhamos como objetivo criar uma coleção de amostras de sarcoma no Biobanco do iMM-CAML, bem como caracterizar metabolicamente estas amostras, utilizando espectroscopia de RMN e avaliação da expressão genética por PCR quantitativo. Os resultados preliminares obtidos por RMN apresentaram diferenças evidentes entre o tecido tumoral e o tecido saudável adjacente. As amostras tumorais indicaram uma alta atividade glicolítica, apresentando níveis elevados de lactato acompanhados por níveis reduzidos de glucose. Além disso, foram demonstrados níveis elevados de glutamato juntamente com níveis mais baixos de glutamina, o que sugere um metabolismo de glutamina mais elevado nestas amostras. Outras diferenças observadas envolvem níveis de glucose-1-fosfato e UDP-glucose, bem como BCAA, aminoácidos aromáticos e carnosina. No que diz respeito à expressão genética, os resultados obtidos mostraram uma expressão mais elevada de LDHA, seguida por GAPDH e ALDOC. A GLS2, envolvida no metabolismo da glutamina, mostra o nível mais baixo de expressão entre os genes avaliados. A continuação da caracterização metabolómica de amostras de STS permitirá o estabelecimento de diferenças discriminativas entre STS e tecido normal, contribuindo para o desenvolvimento de terapias mais eficazes para estes tumores.Sarcomas are a heterogenous group of malignant tumors that originate from the transformation of mesenchymal cells in connective tissues. These account for less than 1% of adult cancers and encompass over 170 subtypes, of which approximately 87% arise from soft tissue and 13% from bone. Carcinogenesis is dependent on alterations of the metabolic activity and presents certain characteristics common to a wide spectrum of different cancers, such as apoptosis evasion, unlimited replicative potential, reprogramming of energy metabolism, among others. Two metabolic substrates commonly used by cancer cells to meet the biosynthetic and bioenergetic requirements of these cells are glucose and glutamine. One way to study the metabolic characteristics of tumors, including sarcomas, comprises several techniques that will be described in this thesis. The resulting Metabolomics is a broad quantitative and qualitative analysis of the metabolome and one of the main advantages regarding this approach lies on the fact that metabolites are downstream of genes, transcripts, and proteins and therefore, give the closest representation of the phenotype. In this project, we aimed to create a collection of sarcoma samples at the biobank of iMM-CAML, as well as metabolically characterize these samples using NMR spectroscopy and evaluation of genetic expression by quantitative PCR. The preliminary results obtained on 1H NMR profiling of STS displayed evident differences between tumoral tissue and adjacent non-involved healthy tissue. Tumor samples indicated high glycolytic activity by displaying higher levels of lactate alongside lower levels of glucose. Furthermore, increased levels of glutamate were shown together with lower levels of glutamine, which suggests a higher glutamine metabolism in these samples. Other prominent differences observed regard levels of glucose-1-phosphate and UDP-glucose as well as BCAA, aromatic amino acids and carnosine. Regarding gene expression, results obtained showed a higher expression of LDHA, followed by GAPDH and ALDOC. GLS2, involved in glutamine metabolism, shows the lowest level of expression amongst the assessed genes. The extension of metabolomic characterization of STS samples will allow the establishment of discriminative differences between STS and normal tissue, contributing to the development of more effective therapies for these tumors.iMM- Instituto de Medicina Molecular João Lobo Antunes

Study of deferiprone-induced agranulocytosis in β-thalassemic patients through a metabolomics approach

ABSTRACT β-Thalassemia is one of the most prevalent forms of congenital blood disorders, characterised by a reduced or absent ability to produce haemoglobin. The mainstay of treatment consists of blood transfusion to maintain the patient’s haemoglobin above 9-10 g/dL. Repeated transfusions result in an excessive accumulation of iron in the body, removal of which is achieved through iron chelating agents. Deferiprone is the first orally bioavailable iron chelator, approved for clinical use in 1997. Considering its potential toxicity, the use of Deferiprone is allowed in Europe only for the treatment of thalassemia major, when Deferoxamine therapy is contraindicated or unappropriated. The main Deferiprone adverse effect is the development of agranulocytosis (in 1–2% of patients). The mechanisms behind this negative effect remain largely unresolved. Currently, only a few metabolomics studies have been performed on neutrophils. Neutrophils are the immune cells forming the major arm of innate immunity. Due to their physiological characteristic, the study of these cells in vitro presents several difficulties meaning that standard protocols for most assays must be optimised. This thesis aimed to establish protocols for the study of the metabolomic profile of human neutrophils in healthy and pathological conditions. In particular, this study aimed to investigate the PMNs (polymorphonuclear leukocytes) metabolic profile of beta thalassemic patients treated with iron chelator Deferiprone, to explore its potential relationship with the onset of agranulocytosis, using a metabolomics approach. Our data demonstrated that analysis of the metabolomic profiles in PMNs with GC-MS, allowed us to identify different metabolites including organic acids, amino acids, fatty acids, and sugars. The results showed a different metabolomic profile between PMNs obtained from patients with Deferiprone-induced agranulocytosis and PMNs of patients without Deferiprone-induced agranulocytosis. Multivariate statistical analysis of GCMS data revealed that the PMNs of patients with Deferiprone-induced agranulocytosis have a metabolic profile characterized by an increase of metabolites directly involved in the metabolic pathways of glutathione synthesis and by a decrease of arachidonic acid stearic acid and inosine. 5 Considering the important physiological roles of these metabolites, our results could shed light on the physiopathological mechanisms of this harmful side effect of DFP treatment. This work is a pilot study that has been only validated in a small independent cohort and, therefore, further confirmation in larger studies is required

RA-MAP, molecular immunological landscapes in early rheumatoid arthritis and healthy vaccine recipients

Rheumatoid arthritis (RA) is a chronic inflammatory disorder with poorly defined aetiology characterised by synovial inflammation with variable disease severity and drug responsiveness. To investigate the peripheral blood immune cell landscape of early, drug naive RA, we performed comprehensive clinical and molecular profiling of 267 RA patients and 52 healthy vaccine recipients for up to 18 months to establish a high quality sample biobank including plasma, serum, peripheral blood cells, urine, genomic DNA, RNA from whole blood, lymphocyte and monocyte subsets. We have performed extensive multi-omic immune phenotyping, including genomic, metabolomic, proteomic, transcriptomic and autoantibody profiling. We anticipate that these detailed clinical and molecular data will serve as a fundamental resource offering insights into immune-mediated disease pathogenesis, progression and therapeutic response, ultimately contributing to the development and application of targeted therapies for RA

RA-MAP, molecular immunological landscapes in early rheumatoid arthritis and healthy vaccine recipients

Rheumatoid arthritis (RA) is a chronic inflammatory disorder with poorly defined aetiology characterised by synovial inflammation with variable disease severity and drug responsiveness. To investigate the peripheral blood immune cell landscape of early, drug naive RA, we performed comprehensive clinical and molecular profiling of 267 RA patients and 52 healthy vaccine recipients for up to 18 months to establish a high quality sample biobank including plasma, serum, peripheral blood cells, urine, genomic DNA, RNA from whole blood, lymphocyte and monocyte subsets. We have performed extensive multi-omic immune phenotyping, including genomic, metabolomic, proteomic, transcriptomic and autoantibody profiling. We anticipate that these detailed clinical and molecular data will serve as a fundamental resource offering insights into immune-mediated disease pathogenesis, progression and therapeutic response, ultimately contributing to the development and application of targeted therapies for RA.</p

MSCs Conditioned Media and Umbilical Cord Blood Plasma Metabolomics and Composition.

Human mesenchymal stem cells (hMSCs) from umbilical cord (UC) blood (UCB) and matrix are tested clinically for a variety of pathologies but in vitro expansion using culture media containing fetal bovine serum (FBS) is essential to achieve appropriate cell numbers for clinical use. Human UCB plasma (hUCBP) can be used as a supplement for hMSCs culture, since UCB is rich in soluble growth factors and due to worldwide increased number of cryopreserved UCB units in public and private banks, without the disadvantages listed for FBS. On the other hand, the culture media enriched in growth factors produced by these hMSCs in expansion (Conditioned medium--CM) can be an alternative to hMSCs application. The CM of the hMSCs from the UC might be a better therapeutic option compared to cell transplantation, as it can benefit from the local tissue response to the secreted molecules without the difficulties and complications associated to the engraftment of the allo- or xeno-transplanted cells. These facts drove us to know the detailed composition of the hUCBP and CM, by 1H-NMR and Multiplexing LASER Bead Technology. hUCBP is an adequate alternative for the FBS and the CM and hUCBP are important sources of growth factors, which can be used in MSCs-based therapies. Some of the major proliferative, chemotactic and immunomodulatory soluble factors (TGF-β, G-CSF, GM-CSF, MCP-1, IL-6, IL-8) were detected in high concentrations in CM and even higher in hUCBP. The results from 1H-NMR spectroscopic analysis of CM endorsed a better understanding of hMSCs metabolism during in vitro culture, and the relative composition of several metabolites present in CM and hUCBP was obtained. The data reinforces the potential use of hUCBP and CM in tissue regeneration and focus the possible use of hUCBP as a substitute for the FBS used in hMSCs in vitro culture

Effect of food extracts and bioactive food compounds on the mechanism of atherosclerosis and nutritional biomarkers

Primera, estudiar los efectos de un extracto de cacahuete rico en polifenoles y de compuestos bioactivos (alfa-tocoferol) en modelos celulares a nivel de inflamación (células monocíticas THP-1) y disfunción endotelial (células endoteliales de aorta humana; HAEC), respectivamente. Segunda, optimizar el volumen sanguíneo caracterizando el perfil de metabolitos acuosos y lipídicos, la composición de ácidos grasos, la detección de subclases de lipoproteínas y de polifenoles en plasma y glóbulos rojos. El extracto de cacahuete ejerce efectos anti-inflamatorios mediante la inhibición de la proteína del TNF-α extracelular, a través de la inhibición de la activación del factor de transcripción c-Jun. El alfa-tocoferol mejora la función endotelial mediante la inhibición de la VCAM-1 y en menor grado sobre la E-selectina e ICAM-1. El plasma y glóbulos rojos aportan información metabolómica complementaria y se elegirá uno u otro en función del objetivo de los estudios en humanos.The thesis addresses two major issues. Firstly: The study the effects of polyphenol-rich peanut extract and bioactive compounds (alpha-tocopherol) in cellular models of inflammation (monocytic cells; THP-1) and on endothelial dysfunction (human aortic endothelial cells; HAEC), respectively. Secondly: The optimisation of blood sampling for human studies to characterise the profile of aqueous and lipid metabolites, fatty acid composition, lipoprotein subclasses, and polyphenol content of plasma and red blood cells. Peanut extract exerts anti-inflammatory effects by inhibiting extracellular TNF-α protein via the inhibition of c-Jun transcription factor activation. Alpha-tocopherol improves endothelial function by inhibiting VCAM-1 and, to a lesser extent, E-selectin and ICAM-1. Analyses of metabolites in plasma and red blood cells generate complementary information. The measurements may need to be performed in either, or both, matrices, depending on the objectives of the study