Non-viral nitric oxide-based gene therapy improves perfusion and liposomal doxorubicin sonopermeation in neuroblastoma models.

Neuroblastoma (NB) is a pediatric malignancy that accounts for 15% of cancer-related childhood mortality. High-risk NB requires an aggressive chemoradiotherapy regimen that causes significant off-target toxicity. Despite this invasive treatment, many patients either relapse or do not respond adequately. Recent studies suggest that improving tumor perfusion can enhance drug accumulation and distribution within the tumor tissue, potentially augmenting treatment effects without inflicting systemic toxicity. Accordingly, methods that transiently increase tumor perfusion prior to treatment may help combat this disease. Here, we show the use of gene therapy to confer inducible nitric oxide synthase (iNOS) expression solely in the tumor space, using focused ultrasound targeting. NOS catalyzes the reaction that generates nitric oxide (NO), a potent endogenous vasodilator. This study reports the development of a targeted non-viral image-guided platform to deliver iNOS-expressing plasmid DNA (pDNA) to vascular endothelial cells encasing tumor blood vessels. Following transfection, longitudinal quantitative contrast-enhanced ultrasound (qCEUS) imaging revealed an increase in tumor perfusion over 72 h, attributed to elevated intratumoral iNOS expression. Methods: To construct a gene delivery vector, cationic ultrasound-responsive agents (known as "microbubbles") were employed to carry pDNA in circulation and transfect tumor vascular endothelial cells in vivo using focused ultrasound (FUS) energy. This was followed by liposomal doxorubicin (L-DOX) treatment. The post-transfection tumor response was monitored longitudinally using qCEUS imaging to determine relative changes in blood volumes and perfusion rates. After therapy, ex vivo analysis of tumors was performed to examine the bioeffects associated with iNOS expression. Results: By combining FUS therapy with cationic ultrasound contrast agents (UCAs), we achieved selective intratumoral transfection of pDNA encoding the iNOS enzyme. While transitory, the degree of expression was sufficient to induce significant increases in tumoral perfusion, to appreciably enhance the chemotherapeutic payload and to extend survival time in an orthotopic xenograft model. Conclusion: We have demonstrated the ability of a novel targeted non-viral gene therapy strategy to enhance tumor perfusion and improve L-DOX delivery to NB xenografts. While our results demonstrate that transiently increasing tumor perfusion improves liposome-encapsulated chemotherapeutic uptake and distribution, we expect that our iNOS gene delivery paradigm can also significantly improve radio and immunotherapies by increasing the delivery of radiosensitizers and immunomodulators, potentially improving upon current NB treatment without concomitant adverse effects. Our findings further suggest that qCEUS imaging can effectively monitor changes in tumor perfusion in vivo, allowing the identification of an ideal time-point to administer therapy.

Histotripsy induces apoptosis and reduces hypoxia in a neuroblastoma xenograft model.

BACKGROUND: Neuroblastoma (NB) is the most common extracranial solid tumor of childhood, and high-risk disease is resistant to intensive treatment. Histotripsy is a focused ultrasound therapy under development for tissue ablation via bubble activity. The goal of this study was to assess outcomes of histotripsy ablation in a xenograft model of high-risk NB. METHODS: Female NCr nude mice received NGP-luciferase cells intrarenally. Under ultrasound image guidance, histotripsy pulses were applied over a distance of 4-6 mm within the tumors. Bioluminescence indicative of tumor viability was quantified before, immediately after, and 24 h after histotripsy exposure. Tumors were immunostained to assess apoptosis (TUNEL), endothelium (endomucin), pericytes (αSMA), hypoxia (pimonidazole), vascular endothelial growth factor A (VEGFA), and platelet-derived growth factor-B (PDGF-B). The apoptotic cytokine TNFα and its downstream effector cleaved caspase-3 (c-casp-3) were assessed with SDS-PAGE. RESULTS: Histotripsy induced a 50% reduction in bioluminescence compared to untreated controls, with an absence of nuclei in the treatment core surrounded by a dense rim of TUNEL-positive cells. Tumor regions not targeted by histotripsy also showed an increase in TUNEL staining density. Increased apoptosis in histotripsy samples was consistent with increases in TNFα and c-casp-3 relative to controls. Treated tumors exhibited a decrease in hypoxia, VEGF, PDGF-B, and pericyte coverage of vasculature compared to control samples. Further, increases in vasodilation were found in histotripsy-treated specimens. CONCLUSIONS: In addition to ablative effects, histotripsy was found to drive tumor apoptosis through intrinsic pathways, altering blood vessel architecture, and reducing hypoxia.

TET2 promotes anti-tumor immunity by governing G-MDSCs and CD8+ T-cell numbers.

The host immune response is a fundamental mechanism for attenuating cancer progression. Here we report a role for the DNA demethylase and tumor suppressor TET2 in host anti-tumor immunity. Deletion of Tet2 in mice elevates IL-6 levels upon tumor challenge. Elevated IL-6 stimulates immunosuppressive granulocytic myeloid-derived suppressor cells (G-MDSCs), which in turn reduce CD8+ T cells upon tumor challenge. Consequently, systematic knockout of Tet2 in mice leads to accelerated syngeneic tumor growth, which is constrained by anti-PD-1 blockade. Removal of G-MDSCs by the anti-mouse Ly6g antibodies restores CD8+ T-cell numbers in Tet2-/- mice and reboots their anti-tumor activity. Importantly, anti-IL-6 antibody treatment blocks the expansion of G-MDSCs and inhibits syngeneic tumor growth. Collectively, these findings reveal a TET2-mediated IL-6/G-MDSCs/CD8+ T-cell immune response cascade that safeguards host adaptive anti-tumor immunity, offering a cell non-autonomous mechanism of TET2 for tumor suppression.

The fusiform gyrus exhibits an epigenetic signature for Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is the most common type of dementia, and patients with advanced AD frequently lose the ability to identify family members. The fusiform gyrus (FUS) of the brain is critical in facial recognition. However, AD etiology in the FUS of AD patients is poorly understood. New analytical strategies are needed to reveal the genetic and epigenetic basis of AD in FUS. RESULTS: A complex of new analytical paradigms that integrates an array of transcriptomes and methylomes of normal controls, AD patients, and "AD-in-dish" models were used to identify genetic and epigenetic signatures of AD in FUS. Here we identified changes in gene expression that are specific to the FUS in brains of AD patients. These changes are closely linked to key genes in the AD network. Profiling of the methylome (5mC/5hmC/5fC/5caC) at base resolution identified 5 signature genes (COL2A1, CAPN3, COL14A1, STAT5A, SPOCK3) that exhibit perturbed expression, specifically in the FUS and display altered DNA methylome profiles that are common across AD-associated brain regions. Moreover, we demonstrate proof-of-principle that AD-associated methylome changes in these genes effectively predict the disease prognosis with enhanced sensitivity compared to presently used clinical criteria. CONCLUSIONS: This study identified a set of previously unexplored FUS-specific AD genes and their epigenetic characteristics, which may provide new insights into the molecular pathology of AD, attributing the genetic and epigenetic basis of FUS to AD development.