The role of alcohol treated-extracellular vesicles in oral cells transformation

• Grant number: 18/18496-6
• Support Opportunities: Research Projects - Thematic Grants
• Start date: April 01, 2020
• End date: March 31, 2026
• Field of knowledge: Health Sciences - Medicine
• Principal Investigator: Adriana Franco Paes Leme
• Grantee: Adriana Franco Paes Leme
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia e Inovação (Brasil). Campinas , SP, Brazil
• Pesquisadores principais: Luiz Paulo Kowalski
• Associated researchers: Alan Roger dos Santos Silva ; Ana Carolina Prado Ribeiro e Silva ; Ângela Saito ; Cesar Andres Rivera Martinez ; Daniela Campos Granato ; Diana Noronha Nunes ; Fabio Albuquerque Marchi ; Fernanda Miriane Bruni Soliani ; Guilherme Pimentel Telles ; Jay William Fox ; Jens Stein ; Luciana Nogueira de Sousa Andrade ; Luiz Paulo Kowalski ; Marcio Ajudarte Lopes ; Marcio Chaim Bajgelman ; Mariane Tami Amano ; Nicholas Sherman ; Roger Chammas ; Rosane Minghim ; Roxane Maria Fontes Piazza ; Sandra Martha Gomes Dias ; Thomas Kislinger
• Associated research grant(s): 23/14874-4 - Single-cell proteomics analyses of immune cells from Head and Neck Cancer patients, AP.R SPRINT; 22/03695-9 - Alcohol can modify antigen processing and presentation through extracellular vesicles that favor immune evasion, AP.R
• Associated scholarship(s): 23/18374-6 - Development of methodologies for protein enrichment in liquid biopsies using functionalized magnetic nanoparticles., BP.MS; 21/14035-7 - Study of extracellular vesicles from Squamous Cell Carcinoma in the modulation of the immune response of macrophages and dendritic cells and their communication with the tumor microenvironment, BP.DD; 21/06125-6 - Structural proteomics strategies to characterize splicing elements as potential therapeutic targets for head and neck squamous cell carcinoma., BP.DR; + associated scholarships 23/12076-3 - Single-cell proteomics applied to the study of blood cells from head and neck cancer patients, BP.DR; 23/08471-4 - Purification and top-down proteomic analysis of PD-1 (programmed cell death 1 protein) from CD8+ T lymphocytes of patients with head and neck cancer, BP.PD; 22/14348-8 - Structural proteomics for the search of therapeutic targets in oral cancer, BP.DR; 22/12815-8 - Combining omics strategies can indicate potential targets involved in the oral cancer initiation through extracellular vesicle communication, BP.PD; 22/13619-8 - Investigate the effect of blocking alcohol-altered proteins from extracellular vesicles on the immune response, BP.PD; 22/04490-1 - Validation of markers of malignant transformation into oral squamous cell carcinoma using imaging mass cytometry and targeted proteomics, BP.DR; 21/03319-4 - Characterization of the role of tumor extracellular vesicles in tolerant tumor microenvironment through TCD4 lymphocyte modulation in oral carcinoma, BP.PD; 21/08034-8 - Study of the effect of extracellular vesicles from oral carcinoma models on neutrophil modulation, BP.DR; 21/05965-0 - Study of extracellular vesicles in the initiation of immune response by dendritic cells and T lymphocytes, BP.PD; 19/09692-9 - Validation of target proteins associated with the progression of potentially malignant disorders into Oral Cancer, BP.DR; 20/11709-4 - Combining omics strategies can indicate potential targets involved in the Oral Cancer initiation through extracellular vesicle communication, BP.PD; 18/12194-8 - Immobilization of multiple potential biomarkers, pure and obtained from liquid biopsies, in Metal-Organic Frameworks (MOFs) as biosensors applied in head and neck Cancer diagnosis and prognosis, BP.PD; 18/02180-0 - Study of the glycoprotein composition in the surface of extracellular vesicles of plasma from patients with oral cancer and its correlation to prognosis, BP.PD; 16/19337-3 - Effect of oral cancer cell-derived extracellular vesicles in the macrophages differentiation, BP.PD - associated scholarships

Abstract

Although there have been several advances in research and improvements in oral squamous cell carcinoma (OSCC) treatments, the survival rate at 5 years from diagnosis is still about 50%. The excessive consumption of alcohol is prominent risk factor in oral cancer. The cancer progression does not depend solely on cancer cell-autonomous defects, but also on the dynamic crosstalk among the components of the microenvironment. Although vast attention has been given to the activation of fibroblasts by tumor cells in cancer progression, the so-called cancer-associated fibroblasts (CAFs), the initiation of cancer guided by quiescent or resting fibroblasts is unknown. These cells are recognized sensors of tissue damage with high plasticity and multipotency, and we propose that they can dictate response to exogenous signals by transferring information through shed extracellular vesicles (EVs) to oral squamous cell and other cells from the microenvironment, driving the initial steps of carcinogenesis. Therefore, our reductionist hypotheses are that (1) alcohol alters resting fibroblast and/or keratinocyte-derived EVs, (2) alcohol-altered EVs can modify the cell microenvironment phenotype, (3) the selected EV proteins are candidates as therapeutic targets. To answer the first hypothesis, we plan to characterize the morphology, size, number and composition of alcohol-treated and non-treated fibroblast and keratinocyte-derived EVs by nanotracking analysis, cryo-electron microscopy, miRNA, protein, metabolite and lipid composition. To test the second hypothesis, we will evaluate in vitro the phenotype of oral cells exposed to fibroblasts and keratinocyte-derived EVs in the main hallmarks of cancer and the modulation of the immune cell response, more specifically tissue-resident DCs (Langerhans and interstitial cells), CD8+ and CD4+ T cells, and their major subtypes using multiparametric flow cytometry. In animal models, using orthotopic mouse model and injecting alcohol-treated and non-treated fibroblast and keratinocyte-derived EVs in cervical and popliteal lymph nodes or tongue and, in addition, the dynamics of the interaction between the tissue microenvironment and draining lymph node will be assessed through intravital two-photon microscopy and laser microdissection followed by single-cell RNAseq and MS-based discovery proteomics of primary site and lymph node tissues. Finally, the association of targeted proteins with excessive consumption of alcohol will be verified in human samples using VEs from biopsy of oral potentially malignant lesions (leukoplakia) and early stage oral squamous cell carcinoma (T1/2N0 and T1/2N+) and their respective plasma VEs, compared with healthy subjects, by using MS-based targeted proteomics. In the third hypothesis as proof-of-concept, we plan strategies based on antibody blocking and cell knockdown against candidates as therapeutic targets revisiting the in vitro and animal models. Herein, we expect that resting fibroblasts and/or keratinocytes are involved in the initial oral squamous cell transformation through extracellular vesicles and indicate potential therapeutic candidates associated with oral cancer initiation. (AU)

Scientific publications

(14)

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

SA, JAMILE DE OLIVEIRA; TRINO, LUCIANA DANIELE; OLIVEIRA, ANA KARINA; BUSSO LOPES, ARIANE FIDELIS; GRANATO, DANIELA CAMPOS; COSTA NORMANDO, ANA GABRIELA; SANTOS, ERISON SANTANA; NEVES, LEANDRO XAVIER; CARNIELLI, CAROLINA MORETTO; PAES LEME, ADRIANA FRANCO. Proteomic approaches to assist in diagnosis and prognosis of oral cancer. EXPERT REVIEW OF PROTEOMICS, v. 18, n. 4, p. 261-284, APR 3 2021. (18/11958-4, 19/21815-9, 18/18496-6, 18/12194-8, 19/18751-9, 19/09692-9, 18/02180-0, 18/15535-0, 18/15728-3, 16/19337-3)

DE CARVALHO-SILVA, LARISSA TINO; NORMANDO, ANA GABRIELA C.; SA, JAMILE DE OLIVEIRA; DOS SANTOS, ERISON SANTANA; DE ROSSI, TATIANE; BUSSO-LOPES, ARIANE FIDELIS; DE OLIVEIRA, ANA KARINA; LEME, ADRIANA F. PAES. Extracellular vesicles in carcinoma microenvironment. BIOCHEMICAL SOCIETY TRANSACTIONS, v. N/A, p. 11-pg., 2023-04-06. (21/08034-8, 22/04490-1, 21/03319-4, 19/09692-9, 21/05965-0, 18/18496-6)

LEME, ADRIANA F. PAES; YOKOO, SAMI; NORMANDO, ANA GABRIELA C.; ORMONDE, JAO VITOR S.; DOMINGUES, ROMENIA RAMOS; CRUZ, FERNANDA F.; SILVA, PEDRO L.; SOUZA, BRUNO S. F.; SANTOS, CLAUDIA C. DOS; CASTRO-FARIA-NETO, HUGO; et al. Proteomics of serum-derived extracellular vesicles are associated with the severity and different clinical profiles of patients with COVID-19: An exploratory secondary analysis. CYTOTHERAPY, v. 26, n. 5, p. 12-pg., 2024-04-30. (19/09692-9, 20/11709-4, 09/54067-3, 18/18496-6)

CARNIELLI, CAROLINA MORETTO; MORAIS, THAYNA MELO DE LIMA; PATRONI, FABIO MALTA DE SA; RIBEIRO, ANA CAROLINA PRADO; BRANDAO, THAIS BIANCA; SOBROZA, EVANDRO; MATOS, LEANDRO LUONGO; KOWALSKI, LUIZ PAULO; LEME, ADRIANA FRANCO PAES; KAWAHARA, REBECA; et al. Comprehensive Glycoprofiling of Oral Tumors Associates N-Glycosylation With Lymph Node Metastasis and Patient Survival. MOLECULAR & CELLULAR PROTEOMICS, v. 22, n. 7, p. 19-pg., 2023-07-01. (18/02180-0, 18/18496-6, 19/17840-8)

BUSSO-LOPES, ARIANE F.; NEVES, LEANDRO X.; CAMARA, GUILHERME A.; GRANATO, DANIELA C.; PRETTI, MARCO ANTONIO M.; HEBERLE, HENRY; PATRONI, FABIO M. S.; SA, JAMILE; YOKOO, SAMI; RIVERA, CESAR; et al. Connecting multiple microenvironment proteomes uncovers the biology in head and neck cancer. NATURE COMMUNICATIONS, v. 13, n. 1, p. 24-pg., 2022-11-07. (19/21815-9, 15/19191-6, 15/50612-8, 09/53998-3, 18/18496-6, 11/00417-3, 09/54067-3, 10/19278-0, 16/07846-0)

BUSSO-LOPES, ARIANE FIDELIS; CARNIELLI, CAROLINA MORETTO; WINCK, FLAVIA VISCHI; DE SA PATRONI, FABIO MALTA; OLIVEIRA, ANA KARINA; GRANATO, DANIELA CAMPOS; PEREIRA E COSTA, RUTE ALVES; DOMINGUES, ROMENIA RAMOS; PAULETTI, BIANCA ALVES; RIANO-PACHON, DIEGO MAURICIO; et al. A Reductionist Approach Using Primary and Metastatic Cell-Derived Extracellular Vesicles Reveals Hub Proteins Associated with Oral Cancer Prognosis. MOLECULAR & CELLULAR PROTEOMICS, v. 20, 2021. (15/19191-6, 18/18496-6, 10/19278-0, 16/07846-0, 19/21815-9)

PAULETTI, BIANCA A.; GRANATO, DANIELA C.; CARNIELLI, CAROLINA M.; CAMARA, GUILHERME A.; NORMANDO, ANA GABRIELA C.; TELLES, GUILHERME P.; LEME, ADRIANA F. PAES. Typic: A Practical and Robust Tool to Rank Proteotypic Peptides for Targeted Proteomics. JOURNAL OF PROTEOME RESEARCH, v. 22, n. 2, p. 7-pg., 2023-02-03. (19/09692-9, 18/02180-0, 19/14828-7, 18/18496-6, 18/15535-0, 19/18751-9)

E COSTA, RUTE A. P.; GRANATO, DANIELA C.; TRINO, LUCIANA D.; YOKOO, SAMI; CARNIELLI, CAROLINA M.; KAWAHARA, REBECA; DOMINGUES, ROMENIA R.; PAULETTI, BIANCA ALVES; NEVES, LEANDRO XAVIER; SANTANA, ALINE G.; et al. ADAM17 cytoplasmic domain modulates Thioredoxin-1 conformation and activity. REDOX BIOLOGY, v. 37, OCT 2020. (16/01528-7, 18/12194-8, 19/18751-9, 18/18496-6, 09/54067-3, 18/15535-0, 14/23888-0, 16/07846-0, 16/24664-3, 14/06485-9, 10/19278-0)

PAULETTI, BIANCA A. .; GRANATO, DANIELA C.; CARNIELLI, CAROLINA M.; CAMARA, GUILHERME A.; NORMANDO, ANA GABRIELA C.; TELLES, GUILHERME P.; LEME, ADRIANA F. PAES. Typic: A Practical and Robust Tool to Rank Proteotypic Peptides for Targeted Proteomics. JOURNAL OF PROTEOME RESEARCH, v. N/A, p. 7-pg., 2022-12-08. (19/18751-9, 19/09692-9, 18/18496-6, 18/15535-0, 19/14828-7)

NEVES, LEANDRO XAVIER; GRANATO, DANIELA C.; BUSSO-LOPES, ARIANE FIDELIS; CARNIELLI, CAROLINA M.; DE SA PATRONI, FABIO M.; DE ROSSI, TATIANE; OLIVEIRA, ANA KARINA; RIBEIRO, ANA CAROLINA P.; BRANDAO, THAIS BIANCA; RODRIGUES, ANDRE NIMTZ; et al. Peptidomics-Driven Strategy Reveals Peptides and Predicted Proteases Associated With Oral Cancer Prognosis. MOLECULAR & CELLULAR PROTEOMICS, v. 20, 2021. (09/54067-3, 18/11958-4, 18/18496-6, 10/19278-0, 16/07846-0)

FIGUEIREDO, DANIELLA; BUSSO-LOPES, ARIANE FIDELIS; DE ROSSI, TATIANE; BERNARDO CRUZ, RODRIGO GOMES; SILVA, JOAO; ORMONDEL, VITOR; SA, JAMILE DE OLIVEIRA; DE CARVALHO SILVA, LARISSA TINO; PAES LEME, ADRIANA FRANCO. Ethanol-treated human fibroblasts and their extracellular vesicles modulate TCD4+lymphocytes. European Journal of Immunology, v. 54, p. 1-pg., 2024-09-01. (18/18496-6, 22/12815-8)

NORMANDO, ANA GABRIELA COSTA; DOS SANTOS, ERISON SANTANA; SA, JAMILE DE OLIVEIRA; BUSSO-LOPES, ARIANE FIDELIS; DE ROSSI, TATIANE; PATRONI, FABIO MALTA DE SA; GRANATO, DANIELA CAMPOS; GUERRA, ELIETE NEVES SILVA; SANTOS-SILVA, ALAN ROGER; LOPES, MARCIO AJUDARTE; et al. A meta-analysis reveals the protein profile associated with malignant transformation of oral leukoplakia. FRONTIERS IN ORAL HEALTH, v. 4, p. 15-pg., 2023-02-27. (22/04490-1, 19/18751-9, 21/03319-4, 19/09692-9, 18/15535-0, 21/05965-0, 18/18496-6)

GRANATO, DANIELA C.; NEVES, LEANDRO X.; TRINO, LUCIANA D.; CARNIELLI, CAROLINA M.; LOPES, ARIANE F. B.; YOKOO, SAMI; PAULETTI, BIANCA A.; DOMINGUES, ROMENIA R.; SA, JAMILE O.; PERSINOTI, GABRIELLA; et al. Meta-omics analysis indicates the saliva microbiome and its proteins associated with the prognosis of oral cancer patients. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS, v. 1869, n. 8, AUG 2021. (18/18496-6, 18/02180-0, 16/07846-0, 19/18751-9, 19/21815-9, 09/54067-3, 18/11958-4, 15/50590-4, 18/12194-8, 18/15728-3, 14/06485-9, 18/15535-0, 10/19278-0)

ZANDONADI, FLAVIA S.; YOKOO, SAMI; GRANATO, DANIELA CAMPOS; RIVERA, CESAR; SOARES MACEDO, CAROLINA CARNEIRO; SOARES, CIRO DANTAS; CARNIELLI, CAROLINA MORETTO; DOMINGUES, ROMENIA RAMOS; PAULETTI, BIANCA A.; CONSONNI, SILVIO ROBERTO; et al. Follistatin-related protein 1 interacting partner of Syndecan-1 promotes an aggressive phenotype on Oral Squamous cell carcinoma (OSCC) models. JOURNAL OF PROTEOMICS, v. 254, p. 13-pg., 2022-03-15. (13/02257-9, 18/18496-6, 16/50005-7, 18/15535-0, 16/07846-0, 19/18751-9, 09/54067-3, 10/19278-0)