Tumoricidal Efficacy of Artesunate-Eluting Microsphere-Based Multimodal Therapy in Patient-Derived Colorectal Tumoroids.

Colorectal cancer (CRC) is a lethal disease characterized by its propensity to metastasize to distant organs. Despite advances in surgery and chemotherapy, CRC remains a major clinical challenge, with high recurrence rates following treatment. The complexity of CRC is further compounded by the limitations of current preclinical models, which often fail to accurately recapitulate the human tumor microenvironment. This underscores the need for improved experimental systems to evaluate novel therapeutic strategies. This study investigates a multimodal second-line treatment strategy using a 3-dimensional (3D), patient-derived CRC tumoroid model that more faithfully mimics the in vivo tumor microenvironment. We evaluated the therapeutic efficacy of a combinatorial approach integrating recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL), artesunate-eluting microspheres (ART-EMs), and mild hyperthermia at 42 °C using a water bath. rhTRAIL selectively induces apoptosis in CRC tumoroids, ART-EMs impose ferroptotic stress, and hyperthermia enhances the crosstalk between these mechanisms. This multitargeted approach is designed to trigger synergistic cell death through the convergence of apoptotic and ferroptotic signaling pathways. Synergistic interactions among rhTRAIL, ART-EMs, and hyperthermia were demonstrated using propidium iodide staining assay, immunoblotting assay, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, JC-1 assay, and dichlorofluorescein assay. Our findings indicate that the multimodal treatment induces greater tumor cell death than individual monotherapies, primarily through amplification of death signaling pathways in tumoroids. The integration of rhTRAIL, ART-EMs, and hyperthermia represents a promising second-line therapeutic strategy for CRC. By harnessing apoptosis-ferroptosis synergy within a clinically relevant 3D model, this approach has the potential to reduce recurrence and improve patient outcomes.