Elucidating the Immunomodulatory Properties of Small Extracellular Vesicles Derived from SMAC Mimetic Treated Cancer Cells

Abstract

Several members of the Inhibitor of Apoptosis (IAP) proteins act as potent anti-apoptotic factors and as key negative regulators of the alternative NF-κB pathway, thereby regulating pro-inflammatory and immune responses. SMAC mimetics (SMs) are a class of drugs designed to antagonize the IAPs, enabling promotion of cell death and stimulation of immune activation. Over the past decade, small extracellular vesicles (sEVs) have gained attention in cancer immunotherapy due to their role in intercellular communication and molecular cargo transfer. This study investigates whether SMs can enhance anti-tumor immune responses mediated by antigen-presenting sEVs. sEVs were isolated from two ovalbumin (OVA)-expressing murine cancer cells, B16F10-OVA and MC38-OVA. The sEVs were characterized using latest MISEV guidelines, such as particle sizing and quantitation, as well as assessment of canonical vesicle-associated markers. The functional capabilities of these sEVs were then evaluated in dendritic cell (DC) co-culture systems to determine their capacity to modulate DC activation, maturation, and antigen presentation. Downstream effects on CD8⁺ T cell responses were assessed using OT-I T cells as a model of recognizing the OVA-specific priming and activation. My findings support a model in which SMs alter tumour sEV release and immune cell activation. These tumour-derived sEVs alongside with SMs promoted DC activation and OVA antigen presentation. Further, the uptake of tumour-derived sEVs within DCs led to activation of antigen-specific CD8⁺ T cells, supporting the role of sEV-mediated transfer of both peptide- and immune-relevant signals. Overall, this work identifies how SMs influence adaptive immunity through the context of sEVs. These findings establish a foundation for further investigation into sEV-mediated mechanisms of anti-tumour immunity and support the therapeutic potential of combining SMs with immune-based strategies in cancer.