Tiana Naylor

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.

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.

Pancreatic ductal adenocarcinoma (PDAC) has quickly risen to become the third leading cause of cancer-related death in the United States. This is in part because of its fibrotic tumor microenvironment (TME) that contributes to poor vascularization and immune infiltration and subsequent chemo- and immunotherapy failure. Here, we investigated an immunotherapy approach combining delivery of stimulator of interferon genes (STING) and Toll-like receptor 4 (TLR4) innate immune agonists by lipid-based nanoparticle (NP) coencapsulation with senescence-inducing RAS-targeted therapies, which can remodel the immune suppressive PDAC TME through the senescence-associated secretory phenotype. Treatment of transplanted and autochthonous PDAC mouse models with these regimens led to enhanced uptake of NPs by multiple cell types in the PDAC TME, induction of type I interferon and other proinflammatory signaling pathways, increased antigen presentation by tumor cells and antigen-presenting cells, and subsequent activation of both innate and adaptive immune responses. This two-pronged approach produced potent T cell–driven and type I interferon–mediated tumor regression and long-term survival in preclinical PDAC models dependent on both tumor and host STING activation. STING and TLR4-mediated type I interferon signaling was also associated with enhanced natural killer and CD8 ⁺ T cell immunity in human PDAC samples. Thus, combining localized immune agonist delivery with systemic tumor-targeted therapy can orchestrate a coordinated type I interferon–driven innate and adaptive immune response with durable antitumor efficacy against PDAC.