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

Grant number: 18/18496-6
Support type:Research Projects - Thematic Grants
Duration: April 01, 2020 - March 31, 2025
Field of knowledge:Health Sciences - Medicine
Principal Investigator:Adriana Franco Paes Leme
Grantee:Adriana Franco Paes Leme
Home Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Ministério da Ciência, Tecnologia, Inovações e Comunicações (Brasil). Campinas , SP, Brazil
Co-Principal Investigators:Luiz Paulo Kowalski
Assoc. researchers:Alan Roger dos Santos Silva ; Ana Carolina Prado Ribeiro e Silva ; Angela 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 ; 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


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)