Multi‐regional Organoid Biobank Reveals FAK‐ACSL1‐Driven Doxorubicin‐Resistance and Predictive Biomarkers in Breast Cancer

ABSTRACT Inter‐tumor and intra‐tumor heterogeneity present significant challenges to achieving precise treatment in breast cancer (BC). To address this, we established a living biobank comprising 68 patient‐derived organoids (PDOs) from 50 patients across various regions, accurately maintaining the histological, genomic, and transcriptomic characteristics of the original tumors. Paired profiling demonstrated high concordance in mutation burden, copy number alterations, and molecular subtypes, capturing both inter‐ and intra‐tumor heterogeneity. Drug screening with four clinically used agents revealed pronounced response variability, enabling the development of multi‐gene expression signatures predictive of drug sensitivity. ACSL1 emerged as a key mediator of Doxorubicin resistance, enriched in malignant epithelial cells exhibiting focal adhesion/MAPK pathway activation and immune‐evasive features. Functional and mechanistic analyses identified focal adhesion kinase (FAK) as an upstream regulator of ACSL1, mediating resistance via ERK and STAT3 signaling. Inhibiting FAK or ACSL1 through pharmacological methods increased the sensitivity of resistant PDOs to Doxorubicin. The combined treatment was effective in decreasing tumor growth and preventing metastasis in xenograft models. Clinically, elevated ACSL1 expression correlated with advanced disease and poor survival. Collectively, these findings define BC heterogeneity, establish prognostic biomarkers, as well as uncover a means of Doxorubicin resistance that supports rational combination therapy.