Haruka Mori

Cellular senescence contributes dynamically to fibrosis and cancer depending on the senescent cell type and its tissue remodeling senescence-associated secretory phenotype; however, current senotherapies lack specificity in targeting pathogenic senescent cells and have limiting toxicities. In a recent issue of Science, Hinterleitner and colleagues designed P-selectin-directed nanoparticles (SMNPs) capable of delivering senotherapies specifically to pathogenic P-selectin+ senescent macrophages in fibrotic tissues while preventing uptake in beneficial senescent endothelial cells. Precision targeting by SMNPs resolved fibrosis and restored organ function in the lung and liver and minimized senotherapeutic toxicity in mice. In tumor models, SMNP targeting of this small senescent macrophage subset was sufficient to overcome fibrosis-mediated immune exclusion and sensitize liver and lung cancers to T cell-directed immunotherapy. P-selectin+ macrophages with senescent features were also enriched in fibrotic human tissues and treatment-refractory cancer patient samples. Overall, this study provides a platform for reducing the toxicity and enhancing the specificity of senotherapies necessary to advance their clinical development for treating chronic disease.

Cellular senescence is a common outcome of cytostatic and cytotoxic cancer therapies. Whereas in some contexts therapy-induced senescence can drive tumor growth suppression and antitumor immunity through the senescence-associated secretory phenotype (SASP), in others it can promote metastatic progression and immune suppression. A systematic interrogation of the impact of different classes of therapy on heterogeneous senescent phenotypes is required to effectively apply these drugs in specific cancer contexts and to rationally design combination treatments. Using genetically defined human and murine prostate cancer cell lines, we found that many classes of cancer therapeutics, including chemotherapies and targeted Aurora kinase and CDK inhibitors, could induce cellular senescence, with DNA-damaging agents mediating the most robust growth arrest and SASP. The SASP produced by chemotherapy-induced senescence, particularly with docetaxel, contained not only inflammatory factors that influence immune responses but also extracellular matrix remodeling factors that facilitate tumor cell dissemination. Though docetaxel treatment could induce senescence and reduce tumor growth in syngeneic prostate cancer mouse models, it was not sufficient to activate antitumor immunity or slow metastatic dissemination. Senolytic therapies targeting the prosurvival BCL2 and IAP pathways upregulated after docetaxel-induced senescence effectively killed senescent tumor cells through multiple cell death pathways, remodeling the inflammatory SASP to repolarize myeloid phenotypes and potentiate CD8+ T-cell activation to further block tumor growth and suppress metastasis. These results suggest that senolytic agents can enhance the immunogenicity and antimetastatic potential of chemotherapy used for treating patients with advanced prostate cancer. Significance: Combining senescence-inducing chemotherapy with senolytic agents can both eliminate senescent cells and enhance the inflammatory output of senescence to drive T-cell immunity and metastasis suppression in prostate cancer.

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.