Role of Glutamine Metabolism in the Metastatic Profile of Circulating Tumor Cells in Breast Cancer

• Grant number: 25/10786-9
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: March 01, 2026
• End date: February 28, 2031
• Field of knowledge: Biological Sciences - Biochemistry - Metabolism and Bioenergetics
• Principal Investigator: Sandra Martha Gomes Dias
• Grantee: Jefferson Felipe Moreira de Sousa
• Host Institution: Centro Nacional de Pesquisa em Energia e Materiais (CNPEM). Campinas , SP, Brazil
• Associated research grant: 21/05726-6 - Metabolism in the microenvironment and the role of metabolic exchanges in tumor progression, AP.TEM

Abstract

Circulating tumor cells (CTCs) represent an aggressive subpopulation of breast cancer cells that play a central role in the establishment of metastases. This project aims to understand how breast cancer CTCs utilize glutamine and to identify vulnerabilities in this metabolic pathway, with the goal of uncovering potential therapeutic targets. To achieve this, we will employ integrated experimental approaches involving chemical inhibition, genetic editing, and both in vitro and in vivo models. Initially, metabolic vulnerabilities will be explored by inhibiting key enzymes involved in glutamine metabolism. The MDA-MB-231 cell line and a patient-derived CTC line (BRx50 Brain), grown under adherent and suspension conditions, will be treated with specific inhibitors. This will be followed by metabolic flux analysis using L-Glutamine (¹³C¿ ¹¿N¿), single-cell transcriptomics (scRNA-Seq), proliferation and viability assays, expression analysis of stemness/mesenchymal markers, and invasion assays. Subsequently, the most relevant targets identified in the initial phase will be validated using targeted gene knockout strategies. Functional and molecular analyses will be repeated to confirm the effects of gene loss. The project will also investigate how changes in glutamine metabolism affect the interaction between CTCs and the immune system, particularly with Natural Killer (NK) cells. Co-culture assays of NK cells with genetically edited CTCs will be conducted to assess immune activation in response to distinct tumor metabolic states. Finally, the metastatic potential of genetically manipulated CTCs will be evaluated in murine models. Immunodeficient NSG mice (lacking NK cells) and Nude mice (retaining NK cells) will be used to investigate the role of NK cells in limiting tumor dissemination. This multidimensional approach will enable the elucidation of key mechanisms linking glutamine metabolism, immune evasion, and metastasis in CTCs, with potential for the identification of novel therapeutic targets. (AU)