Patient-Derived Organoids to Functionally Characterize Chemotherapy Resistance in Breast Cancer

Breast cancer is one of the most common malignancies worldwide, and neoadjuvant chemotherapy (NACT) is a standard treatment approach in patients with locally advanced disease. Achieving pathological complete response (pCR) after NACT is strongly associated with improved long-term outcomes. However, significant inter-patient variability in treatment response is observed, even among patients with similar molecular subtypes, highlighting the need for functional approaches to better understand and predict chemotherapy resistance. Current predictive models based on molecular and genomic profiling are limited in their ability to capture the dynamic and functional behavior of tumors. Patient-derived organoid (PDO) models have emerged as a promising platform that preserves tumor architecture, heterogeneity, and biological characteristics, enabling patient-specific functional drug testing in a controlled ex vivo environment. In this prospective observational study, 40 adult female patients diagnosed with locally advanced breast cancer and scheduled to receive neoadjuvant chemotherapy will be enrolled. Tumor tissues obtained during routine diagnostic or therapeutic procedures will be used to establish PDO cultures. No additional invasive procedures will be performed for research purposes. PDO models will be subjected to standardized drug response assays to evaluate chemotherapy sensitivity and resistance. Quantitative endpoints will include dose-response curves (Half-Maximal Inhibitory Concentration (IC50) values), cell viability assays, and apoptotic activity measurements, particularly Caspase-3/7 activation. These functional parameters will be integrated to define resistance phenotypes at the individual patient level. Clinical and pathological response data, including pCR status, will be collected and correlated with PDO-derived functional results to assess the predictive performance of the PDO platform. In parallel, serum samples obtained during routine clinical care will be analyzed for apoptotic biomarkers, and their association with treatment response will be evaluated. In addition, selected Food and Drug Administration (FDA) and European Medicines Agency (EMA) approved small molecules, particularly those not currently used in breast cancer treatment, will be tested on PDO models to investigate their potential to overcome chemotherapy resistance. This approach aims to identify candidate agents for drug repurposing and to generate a functional drug sensitivity profile for each patient. The ultimate goal of this study is to develop a reproducible and clinically relevant functional assay system that integrates ex vivo drug response data with clinical outcomes. This platform may contribute to improved prediction of treatment response, identification of resistance mechanisms, and development of personalized therapeutic strategies in breast cancer.