Drug resistance frequently emerges in breast cancer during chemotherapy, and standalone immunotherapies often yield insufficient results. Therefore, the clinical advancement of combined chemo-immunotherapy strategies is critically needed. However, traditional two-dimensional cell cultures and animal models remain inadequate for reliably assessing the outcomes of such combined treatments. This study presents a straightforward automated multi-well system developed for producing tumor-associated macrophages (TAMs)-immunized breast cancer organoids encapsulated in alginate hydrogels. Using an automated robotic microinjection approach, alginate hydrogels were formed in situ, followed by the introduction of mixed suspensions containing breast cancer cells and TAMs to generate organoids. Breast cancer organoids incorporating TAMs displayed pronounced resistance to epirubicin, yet this effect was effectively reversed by the targeted immunotherapeutic agent PLX3397. Synergistic benefits were systematically examined through multiple schedules of co-administering PLX3397 alongside epirubicin. Complementary RNA sequencing and quantitative polymerase chain reaction analyses were performed to profile gene expression changes under co-treatment conditions, revealing three significantly altered genes—IL6, CD37, and GLS2—that participate in the tumor necrosis factor signaling pathway, PI3K-Akt signaling pathway, and epidermal growth factor receptor tyrosine kinase inhibitor resistance pathway. Overall, these results highlight the capacity of the automated multi-well platform to serve as a viable substitute for conventional bulk culture techniques in the evaluation of chemo-immunotherapy efficacy.
