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Projeto de investigação
Empowering personal health in bladder cancer patients by disclosing medical information hidden on complex urinary proteomes: diagnostic, prognostic and response to therapy
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Proteomic analysis of chromophobe renal cell carcinoma and benign renal oncocytoma biopsies reveals shared metabolic dysregulation
Publication . Carvalho, Luís B.; Jorge, Susana; López-Fernández, Hugo; Lodeiro, Carlos; Dhir, Rajiv; Campos Pinheiro, Luís; Medeiros, Mariana; Santos, Hugo M.; Capelo, José L.; DQ - Departamento de Química; LAQV@REQUIMTE; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); BioMed Central (BMC)
Background: This study investigates the proteomic landscapes of chromophobe renal cell carcinoma (chRCC) and renal oncocytomas (RO), two subtypes of renal cell carcinoma that together account for approximately 10% of all renal tumors. Despite their histological similarities and shared origins, chRCC is a malignant tumor necessitating aggressive intervention, while RO, a benign growth, is often subject to overtreatment due to difficulties in accurate differentiation. Methods: We conducted a label-free quantitative proteomic analysis on solid biopsies of chRCC (n = 5), RO (n = 5), and normal adjacent tissue (NAT, n = 5). The quantitative analysis was carried out by comparing protein abundances between tumor and NAT specimens. Our analysis identified a total of 1610 proteins across all samples, with 1379 (85.7%) of these proteins quantified in at least seven out of ten LC‒MS/MS runs for one renal tissue type (chRCC, RO, or NAT). Results: Our findings revealed significant similarities in the dysregulation of key metabolic pathways, including carbohydrate, lipid, and amino acid metabolism, in both chRCC and RO. Compared to NAT, both chRCC and RO showed a marked downregulation in gluconeogenesis proteins, but a significant upregulation of proteins integral to the citrate cycle. Interestingly, we observed a distinct divergence in the oxidative phosphorylation pathway, with RO showing a significant increase in the number and degree of alterations in proteins, surpassing that observed in chRCC. Conclusions: This study underscores the value of integrating high-resolution mass spectrometry protein quantification to effectively characterize and differentiate the proteomic landscapes of solid tumor biopsies diagnosed as chRCC and RO. The insights gained from this research offer valuable information for enhancing our understanding of these conditions and may aid in the development of improved diagnostic and therapeutic strategies.
How to dissect viral infections and their interplay with the host-proteome by immunoaffinity and mass spectrometry
Publication . Santos, Hugo M.; Carvalho, Luís B.; Lodeiro, Carlos; Martins, Gonçalo; Gomes, Inês L.; Antunes, Wilson D. T.; Correia, Vanessa; Almeida-Santos, Maria M.; Rebelo-de-Andrade, Helena; Matos, António P. A.; Capelo, J. L.; LAQV@REQUIMTE; DQ - Departamento de Química; Elsevier
The capabilities of bioanalytical mass spectrometry to (i) detect and differentiate viruses at the peptide level whilst maintaining high sample throughput and (ii) to provide diagnosis and prognosis for infected patients are presented as a tutorial in this work to aid analytical chemists and physicians to gain insights into the possibilities offered by current high-resolution mass spectrometry technology and bioinformatics. From (i) sampling to sample treatment; (ii) Matrix-Assisted Laser Desorption Ionization- to Electrospray Ionization -based mass spectrometry; and (iii) from clustering to peptide sequencing; a detailed step-by-step guide is provided and exemplified using SARS-CoV-2 Spike Y839 variant and the variant of concern SARS-CoV-2 Alpha (B.1.1.7 lineage), Influenza B, and Influenza A subtypes AH1N1pdm09 and AH3N2.
Empowering personal health in bladder cancer patients by disclosing medical information hidden on complex urinary proteomes: diagnostic, prognostic and response to therapy
Publication . Carvalho, Luís André Botelho de; Capelo Martínez, José; Pinheiro, Luís; Wiśniewski, Jacek
Bladder cancer (BCa), as the most prevalent malignancy within the urinary system,
poses a significant global health challenge, with nearly 600,000 new cases emerging annually
worldwide. This number is alarmingly projected to double by 2040, according to forecasts by
the World Health Organization, spotlighting the urgent need for minimally invasive and cost-
effective diagnostic options to improve patient outcomes.
This doctoral thesis addresses the urgent call for innovative approaches in managing
BCa and potentially other urological cancers. It details the development and application of
cutting-edge proteomic technologies to refine the accuracy of BCa diagnosis and improve
prognostic evaluations. By extending this research to include renal cancers, the thesis under-
scores the proposed methods' versatility and potential broad applicability.
Central to this work is the novel differential personal pathway index (dPPi), a personal-
ized medicine approach derived from extensive work in urine sample processing, proteome
analysis, and mass spectrometry data normalization. This thesis presents several key advance-
ments, including (i) The development of a novel urine protein preservation method using filter-
aided sample preparation (FASP), ensuring effective concentration and stabilization for long-
term storage without compromising sample integrity; (ii) The introduction of a high-through-
put ultrafast proteomics method known as US-FASP, which speeds sample processing while
maintaining robustness and scalability; (iii) The evaluation of normalization techniques for mass
spectrometry data. (iv) Identifying urinary proteomic signatures associated with BCa recur-
rence, highlighting potential prognostic indicators; (v) Implementing longitudinal urine prote-
omic analysis using the dPPi approach, to provide insights into BCa progression over time and
guide clinical interventions; (vi) Exploring proteomic landscapes in chromophobe renal cell car-
cinoma (chRCC) and renal oncocytomas (RO), offering insights into diagnostic and therapeutic
avenues Overall, this thesis contributes significantly to understanding BCa and opens up new
avenues for developing personalized medical interventions. The findings enhance our grasp of
BCa's biological underpinnings and offer a promising framework for extending these innova-
tions to a wider array of urological and other malignancies.
Biochemical network analysis of protein-protein interactions to follow-up T1 bladder cancer patients
Publication . Carvalho, Luís B.; Martínez, José Luis Capelo; Lodeiro, Carlos; Bento, Rafael; Dhir, Rajiv; Morrissey, Jeremiah J.; Pinheiro, Luís Campos; Medeiros, Mariana; Santos, Hugo M.; DQ - Departamento de Química; LAQV@REQUIMTE; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); Elsevier
Bladder cancer (BCa) is a prevalent disease with a high risk of aggressive recurrence in T1-stage patients. Despite the efforts to anticipate recurrence, a reliable method has yet to be developed. In this work, we employed high-resolution mass spectrometry to compare the urinary proteome of T1-stage BCa patients with recurring versus non-recurring disease to uncover actionable clinical information predicting recurrence. All patients were diagnosed with T1-stage bladder cancer between the ages of 51 and 91, and urine samples were collected before medical intervention. Our results suggest that the urinary myeloperoxidase to cubilin ratio could be used as a new tool for predicting recurrence and that dysregulation of the inflammatory and immune systems may be a key driver of disease worsening. Furthermore, we identified neutrophil degranulation and neutrophil extracellular traps (NETs) as key pathways in the progression of T1-stage BCa. We propose that proteomics follow-up of the inflammatory and immune systems may be useful for monitoring the effectiveness of therapy. Significance: This article describes how proteomics can be used to characterize tumor aggressiveness in patients with the same diagnosis of bladder cancer (BCa). LC-MS/MS in combination with label free quantification (LFQ) were used to explore potential protein and pathway level changes related to the aggressiveness of the disease in 13 and 17 recurring and non-recurring T1 stage BCa patients. We have shown that the MPO/CUBN protein ratio is a candidate for a urine prognosis tool in BCa. Furthermore, we identify dysregulation of inflammation process as a driver for BCa recurrence and progression. Moreover, we propose using proteomics to track the effectiveness of therapy in the inflammatory and immune systems.
Pathway-guided monitoring of the disease course in bladder cancer with longitudinal urine proteomics
Publication . Carvalho, Luís Botelho; Capelo, José Luís; Lodeiro, Carlos; Dhir, Rajiv; Pinheiro, Luís Campos; Lopez-Fernandez, Hugo; Martins, Gonçalo; Medeiros, Mariana; Díaz, Fernando; Santos, Hugo Miguel; DQ - Departamento de Química; LAQV@REQUIMTE; NOVA Medical School|Faculdade de Ciências Médicas (NMS|FCM); Springer Nature
Background Monitoring bladder cancer over time requires invasive and costly procedures. Less invasive approaches are required using readily available biological samples such as urine. In this study, we demonstrate a method for longitudinal analysis of the urine proteome to monitor the disease course in patients with bladder cancer. Methods We compared the urine proteomes of patients who experienced recurrence and/or progression (n = 13) with those who did not (n = 17). We identified differentially expressed proteins within various pathways related to the hallmarks of cancer. The variation of such pathways during the disease course was determined using our differential personal pathway index (dPPi) calculation, which could indicate disease progression and the need for medical intervention. Results Seven hallmark pathways are used to develop the dPPi. We demonstrate that we can successfully longitudinally monitor the disease course in bladder cancer patients through a combination of urine proteomic analysis and the dPPi calculation, over a period of 62 months. Conclusions Using the information contained in the patient’s urinary proteome, the dPPi reflects the individual’s course of bladder cancer, and helps to optimise the use of more invasive procedures such as cystoscopy.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
OE
Número da atribuição
SFRH/BD/144222/2019