Michael A. Brehm

Murine research on atherosclerosis has been conducted largely on the C57BL/6 (B6) strain background, and most commonly, the B6.129P2‐ Apoe ^tm1Unc /J (B6‐ApoE ^KO ) strain. The use of the B6 background strain limits translatability for immunotherapy development due to inherent species differences between human and mouse immune systems. A proposed solution to this limitation is a humanized mouse model of atherosclerosis that would support the engraftment of human lymphoid and myeloid cell populations. The development of such a model has been hindered by the lack of a mouse strain that both develops atherosclerosis and supports human immune cell engraftment. In this paper, we report on a NOD.Cg‐ Prkdc ^scid Il2rg ^tm1Wjl /Sz (NSG) immunodeficient mouse strain that has a 383 bp deletion in the Apoe gene which prevents ApoE protein expression. When maintained on a high fat diet, NSG‐ApoE ^KO mice develop hypercholesteremia and atherosclerosis at levels similar to B6‐ApoE ^KO mice. While atherosclerosis in B6‐ApoE ^KO mice has been widely studied in many laboratories under multiple conditions, our initial study of disease development in NSG‐ApoE ^KO mice was limited and future comparative studies with B6‐ApoE ^KO mice are necessary. NSG‐ApoE ^KO mice concomitantly support human cell engraftment while developing atherosclerosis, thereby making them useful to study human‐specific immune aspects.

SUMMARYPancreatic islet-like clusters differentiated from induced pluripotent or embryonic stem cells (SC-islets) are a promising cellular therapy for type 1 diabetes. A standardized and easily applied protocol to assess the quality of SC-islet differentiations would be a valuable resource to ensure consistency across cell preparations. We present a workflow for processing SC-islet clusters to single cells and for intracellular flow cytometry stains using commercially available reagents and antibodies to distinguish cell types generated during a 7-stage differentiation protocol.

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.

Bacterial colonization of tumors is widespread, yet the dynamics during colonization remain underexplored. Here we discover strong variability in the sizes of intratumor bacterial clones and use this variability to infer the mechanisms of colonization. We monitored bacterial population dynamics in murine tumors after introducing millions of genetically barcoded Escherichia coli cells. Results from intravenous injection revealed that roughly a hundred bacteria seeded a tumor and that colonizers underwent rapid, yet highly nonuniform growth. Within a day, bacteria reached a steady-state and then sustained load and clone diversity. Intratumor injections, circumventing colonization bottlenecks, revealed that the nonuniformity persists and that the sizes of bacterial progenies followed a scale-free distribution. Theory suggested that our observations are compatible with a growth model constrained by a local niche load, global resource competition, and noise. Our work provides the first dynamical model of tumor colonization and may allow distinguishing genuine tumor microbiomes from contamination.

Bacterial colonization of tumors is widespread, yet the dynamics during colonization remain underexplored. Here we discover strong variability in the sizes of intratumor bacterial clones and use this variability to infer the mechanisms of colonization. We monitored bacterial population dynamics in murine tumors after introducing millions of genetically barcoded Escherichia coli cells. Results from intravenous injection revealed that roughly a hundred bacteria seeded a tumor and that colonizers underwent rapid, yet highly nonuniform growth. Within a day, bacteria reached a steady-state and then sustained load and clone diversity. Intratumor injections, circumventing infection bottlenecks, revealed that the nonuniformity persists and that the sizes of bacterial progenies followed a scale-free distribution. Theory suggested that our observations are compatible with a growth model constrained by a local niche load, global resource competition, and noise. Our work provides the first dynamical model of tumor colonization and may allow distinguishing genuine tumor microbiomes from contamination.