A comparative analysis of paired normal, primary, and metastatic breast cancer organoids derived from core-needle biopsies

Background: Patient-derived organoids (PDOs) are emerging as clinically relevant ex vivo models that preserve the histology, immunophenotype, and genomic landscape of source tissues. However, generating PDOs from low-abundance core-needle biopsy (CNB) samples, especially paired normal, tumor, and lymph node (LN) tissues, remains challenging. Objective: Previously, we developed an R 3 CE-based culture platform to overcome these limitations and successfully generate paired PDOs from CNB specimens of breast cancer patients. Here we evaluate their fidelity through histopathology and genomic profiling of the R 3 CE-based PDOs. Methods: CNB samples were processed via mincing, enzymatic digestion, and cell isolation, then seeded onto R 3 CE with optimized media. Organoid formation was monitored by bright-field imaging. Growth potential across passages and freeze-thaw stability were also analyzed to evaluate proliferation potential of the established PDOs. Immunohistochemistry (IHC) assessed panCK (AE1/AE3), CD45, Ki67 and breast cancer markers (ER, PR, HER2), with HER2-DISH were used for breast tissue verification. Whole-exome sequencing were applied to evaluated mutation concordance in between various of samples including original tissue. Results: Robust organoid formation was achieved from paired normal, tumor, and LN biopsy specimens. Long-term culture demonstrated stable proliferation and post-thaw viability. Tumor PDOs were panCK-positive/CD45-negative, confirming epithelial origin. Histological concordance between PDOs and original biopsies was HER2 83%, ER 58%, PR 50%, with Ki-67 proliferation preserved. Genomic analysis revealed actionable mutations retained in tumor and LN PDOs but absent in normal PDOs. Conclusions: The R 3 CE platform enables PDO establishment from minimal CNB material, including LN samples, with high histopathological and genomic fidelity. This approach supports PDO biobanking and downstream model evaluations including drug screening for precision oncology.