Comparative Assessment of Various Formats of TRAIL in Three-Dimensional Cell Culture and Patient-Derived Tumoroids of Colorectal Cancer

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising cytokine that selectively targets cancer cells while sparing normal tissues. Despite its favorable safety profile, clinical trials have demonstrated antitumor responses in only a small subset of patients. This limited efficacy has been largely attributed to the short plasma half-life of recombinant monomeric soluble TRAIL (rhTRAIL). To enhance its stability and therapeutic potential, researchers have developed modified versions, including an immunoglobulin Fc domain-fused TRAIL (Fc-TRAIL) and a dimeric Fc-fused single-chain variant (Fc-scTRAIL). In this study, we used the SNU-1746 three-dimensional (3D) multicellular layer culture model and a patient-derived colon cancer tumoroid model to evaluate the biological activity of these TRAIL formats (rhTRAIL, Fc-TRAIL, and Fc-scTRAIL). Treatment with rhTRAIL revealed that a longer exposure time (18-24 hours) was required to induce apoptosis in both 3D models, in contrast to monolayer cultures. Among the TRAIL formats, Fc-scTRAIL was the most potent in inducing apoptosis, as confirmed by immunoblotting analyses. Furthermore, artesunate (ART) enhanced TRAIL-induced apoptosis across all TRAIL formats, with the strongest synergistic effect observed in combination with Fc-scTRAIL. JC-1 staining assays indicated that mitochondrial membrane depolarization (a hallmark of the intrinsic apoptosis pathway) plays a key role in the cell death observed with the combination treatment in tumoroids. These findings provide compelling preclinical evidence supporting the potential of ART and Fc-scTRAIL combination therapy for future clinical evaluation.