Ronnie W. Dinnell

Supplementary Figure 1: NP characterization and long-term responses. (A) NP hydrodynamic size was measured via dynamic light scattering for the indicated NP formulation. NP zeta potential was measured for the indicated NP formulation. (B) Kaplan-Meier survival curve for TH-MYCN model (N=10 mice per group). (C) Whole-mouse BLI measurements were taken at the indicated time point following SH-SY5Y tumor inoculation in hu-NSG mice. Error bars, ± SEM. P values were calculated using 2-way ANOVA. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, n.s, not significant.

Effective treatments for high-risk neuroblastoma, a pediatric malignancy with dismal survival outcomes, demand strategies that remodel the “cold” immunosuppressive tumor microenvironment (TME) to “hot” and proinflammatory. In this study, we interrogate the mechanistic efficacy of a pathogen-inspired nanoparticle (NP) technology that leverages the advantages of lipids to co-deliver a combination of physically distinct but functionally synergistic innate immune agonists for dual pathway activation in humanized mouse models. Specifically, our immunomodulatory NPs (immuno-NPs) co-deliver agonists of the stimulator of interferon (IFN) genes and Toll-like receptor 4 pathways. Together, dual pathway activation in target cells in the TME by immuno-NPs promotes the synergistic production of type I IFNs and other proinflammatory cytokines that powerfully remodel the cytokine milieu. We show that immuno-NPs can be delivered safely in the systemic blood circulation for deposition in the perivascular space of neuroblastoma primary tumors and metastases. Their broad uptake across immune cells, endothelial cells, and even tumor cells drives significant immune activation and restructuring of the cytokine milieu of the TME. We also demonstrate a pivotal role for otherwise exhausted CD8+ T cells that are local and already infiltrating the tumor. Immuno-NP–mediated rescue of this local T-cell subset is chiefly responsible for the acute clearance responses that may be essential for treatment efficacy in aggressive cancers like high-risk neuroblastoma. Taken together, our findings make a strong case for the use of pathogen-inspired, multi-innate pathway-activating therapies, such as immuno-NPs, for high-risk neuroblastoma and other solid tumor malignancies that urgently warrant effective TME remodeling approaches.

Supplementary Figure 3: NP uptake, immune cell proliferation, and activation in FLT3L primary tumor and metastatic liver. (A) Experimental timeline showing injection of SH-SY5Y hu-NSG FLT3L mice with immuno NPs or empty NPs. Created with BioRender.com (B) Flow cytometry analysis of NP uptake in FLT3L primary tumor by CD45+ leukocytes, CD11c+ DCs, CD56+ NK cells, CD8+ T cells, and tumor cells (N=4 mice per group). (C) Flow cytometry analysis of the proliferation of CD45+ leukocytes, CD11c+ DCs, CD56+ NK cells, tumor cells, and CD8+ T cells in the primary tumor (N=4 mice per group). (D) Flow cytometry analysis of activation of CD56+ NK cells and CD8+ T cells in the FLT3L primary tumor via CD69 expression (N=4 mice per group). (E) Flow cytometry analysis of NP uptake in the FLT3L metastatic liver by CD45+ leukocytes, CD11c+ DCs, CD56+ NK cells, tumor cells, and CD8+ T cells (N=4 mice per group). (F) Flow cytometry analysis of proliferation of CD45+ leukocytes, CD11c+ DCs, CD56+ NK cells, tumor cells, and CD8+ T cells in the FLT3L metastatic liver (N=4 mice per group). (G) Flow cytometry analysis of activation of CD56+ NK cells and CD8+ T cells in the FLT3L metastatic liver via CD69 expression (N=4 mice per group). For flow cytometry, technical duplicates were averaged and plotted. Error bars, ± SEM. P values were calculated using 2-way ANOVA. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, n.s, not significant.

Supplementary Figure 2: NP uptake, endothelial cell and macrophage proliferation, cytokine production, and immune cell proliferation. (A-C) Flow cytometry analysis of NP uptake by CD31+ endothelial cells, CD62E+CD31+ endothelial cells, and CD33+ macrophages in the primary tumor (A), metastatic liver (B), and metastatic lungs (C) (N=6 mice per group). (D-F) Proliferation of CD31+ endothelial cells, CD62E+ cells, and CD33+ macrophages in the primary tumor (D), metastatic liver (E), and metastatic lungs (F) (N=6 mice per group). (G-H) Flow cytometry analysis of expression of the indicated cytokine by CD33+ macrophages in the primary tumor (G), metastatic liver (H), and metastatic lungs (I) (N=6 mice per group). (J) Flow cytometry analysis of NP uptake by CD45+ leukocytes, CD11c+ DCs, and CD56+ NK cells in the blood (N=6 mice per group). (K) Proliferation of CD45+ leukocytes, CD11c+ DCs, and CD56+ NK cells in the blood (N=6 mice per group). For flow cytometry, technical duplicates were averaged and plotted. Error bars, ± SEM. P values were calculated using 2-way ANOVA. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05, n.s, not significant.

Immunotherapy has limited success in pancreatic ductal adenocarcinoma (PDAC) due to an immune exclusive tumor microenvironment (TME) that lacks many cytokines necessary for Natural Killer (NK) and T cell responses. Here, we design multiplexed mRNAs encoding interleukins, chemokines, and interferons as a safe and effective cytokine therapy for PDAC. Intratumoral injection of IL-12, IL-18, CCL5, CXCL10, and IFNβ mRNAs achieves robust yet transient cytokine expression, leading to NK and CD8⁺ T cell activation and reduced tumor growth and fibrosis in PDAC transplant mouse models. Combining cytokine with tumor antigen mRNAs enhances dendritic cell antigen presentation and CD8⁺ T cell priming locally and systemically that prolongs animal survival after a single dose. Remarkably, nanoparticle encapsulation of the cytokine/antigen mRNA cocktail allows systemic administration and local delivery to autochthonous PDAC tumors in mice, culminating in curative responses in 50% of animals and antigen-reactive T cell persistence. These results suggest that multiplexed mRNA approaches to deliver cytokines and antigens generally absent in the TME could pave the way for effective immunotherapy in PDAC.

Effective treatments for high-risk neuroblastoma, a pediatric malignancy with dismal survival outcomes, demand strategies that remodel the “cold” immunosuppressive tumor microenvironment (TME) to “hot” and proinflammatory. In this study, we interrogate the mechanistic efficacy of a pathogen-inspired nanoparticle (NP) technology that leverages the advantages of lipids to co-deliver a combination of physically distinct but functionally synergistic innate immune agonists for dual pathway activation in humanized mouse models. Specifically, our immunomodulatory NPs (immuno-NPs) co-deliver agonists of the stimulator of interferon (IFN) genes and Toll-like receptor 4 pathways. Together, dual pathway activation in target cells in the TME by immuno-NPs promotes the synergistic production of type I IFNs and other proinflammatory cytokines that powerfully remodel the cytokine milieu. We show that immuno-NPs can be delivered safely in the systemic blood circulation for deposition in the perivascular space of neuroblastoma primary tumors and metastases. Their broad uptake across immune cells, endothelial cells, and even tumor cells drives significant immune activation and restructuring of the cytokine milieu of the TME. We also demonstrate a pivotal role for otherwise exhausted CD8+ T cells that are local and already infiltrating the tumor. Immuno-NP–mediated rescue of this local T-cell subset is chiefly responsible for the acute clearance responses that may be essential for treatment efficacy in aggressive cancers like high-risk neuroblastoma. Taken together, our findings make a strong case for the use of pathogen-inspired, multi-innate pathway-activating therapies, such as immuno-NPs, for high-risk neuroblastoma and other solid tumor malignancies that urgently warrant effective TME remodeling approaches.