• Cancer Stem Cells (CSCs)
• Fluorescence-activated cell sorting (FACS)
• Metabolic plasticity
• Stemness in Breast Cancer
• Tumor heterogeneity

Breast cancer (BC) is a heterogeneous disease with distinct molecular subtypes differing in biological behavior and clinical outcome. A major driver of this heterogeneity is the presence of cancer stem cells (CSCs), endowed with self-renewal and tumor-initiating capacity, contributing to progression, relapse, and therapy resistance.

This project investigated the link between stemness, tumor heterogeneity, and metabolic plasticity in BC using cell lines and patient-derived cultures from primary and metastatic tumors. CSCs were analyzed by flow cytometry and fluorescence-activated cell sorting (FACS) based on CD44/CD24 expression and ATP content, while functional assays (wound healing, colony, spheroid) assessed migration, clonogenicity, and self-renewal.

TNBC models displayed higher CD44⁺/CD24⁻ (CSCs) cells than luminal models, with enhanced migratory and clonogenic ability, supporting the association between stemness and aggressiveness. Metabolic profiling revealed enrichment of CSC-like cells in ATP-low fractions of TNBC, whereas ATP-high fractions showed reduced CD44⁺/CD24⁻ cells, indicating metabolic regulation of stemness, absent in luminal models. In patient-derived cultures, CD44⁺/CD24⁻ cells were rare, while CD44⁺/CD24⁺ and CD44⁻/CD24⁺ populations, enriched in TNBC and bone metastases, correlated with aggressiveness.

Overall, these findings highlight the need to consider heterogeneous CSC-related subpopulations beyond the classical CD44⁺/CD24⁻ profile and suggest CD24 as a marker of aggressiveness in TNBC, reinforcing the interplay between metabolic status and stem-like traits.