Janus-Based Nanopieces for mRNA Delivery Enhance Cancer Immunotherapeutic Efficacy in 3D Sarcoma Organoid Models

Introduction: : Sarcomas are a rare and heterogeneous group of malignant tumors arising from mesenchymal tissues, often characterized by poor prognosis and limited treatment options. Due to their diversity and chemoresistance, developing personalized therapeutic strategies remains a major clinical challenge. Three-dimensional (3D) sarcoma organoids offer a promising platform for modeling tumor heterogeneity and evaluating therapeutic efficacy in a patient-specific manner. On the other hand, interleukin-12 (IL-12) has demonstrated potent anti-tumor properties by stimulating innate and adaptive immune responses; however, its clinical application is limited by systemic toxicity and a short half-life that hampers sustained therapeutic efficacy [1]. To address these challenges, Janus-Based Nanopieces (JBNps) have emerged as next-generation nanocarriers for cancer therapy. These nanoparticles enhance targeting ability, tissue penetration, and enable efficient mRNA delivery due to their rod-shaped, self-assembled nanotubes inspired by DNA base pairing [2]. In this study, we aim to investigate the efficacy of JBNp-mediated IL-12 mRNA delivery in sarcoma organoid models, exploring how nanoparticle architecture modulates release kinetics and prolongs localized cytokine expression to improve anti-tumor activity while minimizing systemic side effect.

Materials and

Methods: : We used functionalized Matrigel as a scaffold to mimic tumor architecture and co-cultured osteosarcoma cell lines with synovial fibroblasts and mesenchymal stem cells, with or without macrophages. Organoids were then co-cultured with T cells in customized media. The organoids were characterized using live/dead cell assays and immunostaining to confirm proper formation. JBNps were fabricated by sonicating self-assembled nanotubes with IL-12 mRNA, resulting in smaller, mRNA-loaded nanostructures. Particle morphology and drug release profiles were characterized using TEM, DLS, and UV-Vis spectroscopy. Organoids were incubated with JBNps to assess IL-12 mRNA deliver, cytotoxicity and immune responses.

Results, Conclusions, and Discussions:: Sarcoma organoids can be quickly generated, with diameters ranging from 1000 to 1500 µm, exhibiting a spherical shape and compact structure. The organoid models maintained high cell viability throughout the culture period, suggesting strong in vitro reliability. We also demonstrated an immune-featured organoid model and its method for testing personalized immune therapeutic responses. JBNps demonstrate enhanced penetration and retention within sarcoma organoids compared to free mRNA. JBNps successfully delivered IL-12 mRNA into cells, resulting in cytokine expression and secretion into the tumor microenvironment. Importantly, JBNps showed a higher overall IL-12 expression level and a more homogeneous delivery profile than lipid nanoparticles (LNPs), highlighting their superior delivery efficiency. Our findings also indicated that JBNps can overcome the delivery barriers presented by dense tumor matrices, offering both spatially controlled drug distribution and reduced systemic stress. This approach is particularly advantageous in sarcoma treatment, where conventional therapies often fail due to heterogeneous vasculature and high interstitial pressure. In addition to enhanced delivery, IL-12 expression induced by JBNps significantly modulated the immune microenvironment within the organoids. We observed increased infiltration and activation of CD8⁺ T cells, maturation of dendritic cells, macrophage repolarization, upregulation of interferon-γ, and reduced expression of immunosuppressive markers such as PD-L1, indicating a shift from a “cold tumor” to a “hot tumor” microenvironment. Furthermore, by integrating immune components into the organoid platform, we provide a more accurate and translatable system for testing immunotherapies in a personalized manner, overcoming limitations of conventional 2D cultures and immunodeficient animal models.

Overall, the immune-featured sarcoma organoid model provides a physiologically relevant in vitro platform for preclinical drug evaluation. JBNp-mediated mRNA delivery represents a promising approach for advancing precision nanomedicine, particularly in the treatment of rare and treatment-resistant cancer such as sarcomas.

Acknowledgements and/or References (Optional):: 1. Landoni, E., et al., IL-12 reprograms CAR-expressing natural killer T cells to long-lived Th1-polarized cells with potent antitumor activity. Nature Communications, 2024. 15(1): p. 89.
2. Lee, J., et al., DNA-inspired nanomaterials for enhanced endosomal escape. Proceedings of the National Academy of Sciences USA, 2021. 118(19).