Meijing Wu

Cell-Penetrating Peptide-Modified Gold Nanoparticles for the Delivery of Doxorubicin to Brain Metastatic Breast Cancer

As therapies continue to increase the lifespan of patients with breast cancer, the incidence of brain metastases has steadily increased, affecting a significant number of patients with metastatic disease. However, a major barrier toward treating these lesions is the inability of therapeutics to penetrate into the central nervous system and accumulate within intracranial tumor sites. In this study, we designed a cell-penetrating gold nanoparticle platform to increase drug delivery to brain metastatic breast cancer cells. TAT peptide-modified gold nanoparticles carrying doxorubicin led to improved cytotoxicity toward two brain metastatic breast cancer cell lines with a decrease in the IC50 of at least 80% compared to free drug. Intravenous administration of these particles led to extensive accumulation of particles throughout diffuse intracranial metastatic microsatellites with cleaved caspase-3 activity corresponding to tumor foci. Furthermore, intratumoral administration of these particles improved survival in an intracranial MDA-MB-231-Br xenograft mouse model. Our results demonstrate the promising application of gold nanoparticles for improving drug delivery in the context of brain metastatic breast cancer.

CCL2 Produced by the Glioma Microenvironment Is Essential for the Recruitment of Regulatory T Cells and Myeloid-Derived Suppressor Cells

In many aggressive cancers, such as glioblastoma multiforme, progression is enabled by local immunosuppression driven by the accumulation of regulatory T cells (Treg) and myeloid-derived suppressor cells (MDSC). However, the mechanistic details of how Tregs and MDSCs are recruited in various tumors are not yet well understood. Here we report that macrophages and microglia within the glioma microenvironment produce CCL2, a chemokine that is critical for recruiting both CCR4+ Treg and CCR2+Ly-6C+ monocytic MDSCs in this disease setting. In murine gliomas, we established novel roles for tumor-derived CCL20 and osteoprotegerin in inducing CCL2 production from macrophages and microglia. Tumors grown in CCL2-deficient mice failed to maximally accrue Tregs and monocytic MDSCs. In mixed-bone marrow chimera assays, we found that CCR4-deficient Treg and CCR2-deficient monocytic MDSCs were defective in glioma accumulation. Furthermore, administration of a small-molecule antagonist of CCR4 improved median survival in the model. In clinical specimens of glioblastoma multiforme, elevated levels of CCL2 expression correlated with reduced overall survival of patients. Finally, we found that CD163-positive infiltrating macrophages were a major source of CCL2 in glioblastoma multiforme patients. Collectively, our findings show how glioma cells influence the tumor microenvironment to recruit potent effectors of immunosuppression that drive progression. Cancer Res; 76(19); 5671-82. ©2016 AACR.

RhoA Kinase Inhibition With Fasudil Versus Simvastatin in Murine Models of Cerebral Cavernous Malformations.

Background and Purpose— We sought to compare the effect of chronic treatment with commonly tolerated doses of Fasudil, a specific RhoA kinase (ROCK) inhibitor, and simvastatin (with pleiotropic effects including ROCK inhibition) on cerebral cavernous malformation (CCM) genesis and maturation in 2 models that recapitulate the human disease. Methods— Two heterozygous murine models, Ccm1+/−Msh2−/− and Ccm2+/−Trp53−/−, were treated from weaning to 4 to 5 months of age with Fasudil (100 mg/kg per day), simvastatin (40 mg/kg per day) or with placebo. Mouse brains were blindly assessed for CCM lesion burden, nonheme iron deposition (as a quantitative measure of chronic lesional hemorrhage), and ROCK activity. Results— Fasudil, but not simvastatin, significantly decreased mature CCM lesion burden in Ccm1+/−Msh2−/− mice, and in meta-analysis of both models combined, when compared with mice receiving placebo. Fasudil and simvastatin both significantly decreased the integrated iron density per mature lesion area in Ccm1+/−Msh2−/− mice, and in both models combined, compared with mice given placebo. ROCK activity in mature lesions of Ccm1+/−Msh2−/− mice was similar with both treatments. Fasudil, but not simvastatin, improved survival in Ccm1+/−Msh2−/− mice. Fasudil and simvastatin treatment did not affect survival or lesion development significantly in Ccm2+/−Trp53−/− mice alone, and Fasudil benefit seemed limited to males. Conclusions— ROCK inhibitor Fasudil was more efficacious than simvastatin in improving survival and blunting the development of mature CCM lesions. Both drugs significantly decreased chronic hemorrhage in CCM lesions. These findings justify the development of ROCK inhibitors and the clinical testing of commonly used statin agents in CCM.

Mutant IDH1 and seizures in patients with glioma

Objective: Because the d-2-hydroxyglutarate (D2HG) product of mutant isocitrate dehydrogenase 1 (IDH1mut) is released by tumor cells into the microenvironment and is structurally similar to the excitatory neurotransmitter glutamate, we sought to determine whether IDH1mut increases the risk of seizures in patients with glioma, and whether D2HG increases the electrical activity of neurons. Methods: Three WHO grade II-IV glioma cohorts from separate institutions (total N = 712) were retrospectively assessed for the presence of preoperative seizures and tumor location, WHO grade, 1p/19q codeletion, and IDH1mut status. Rat cortical neurons were grown on microelectrode arrays, and their electrical activity was measured before and after treatment with exogenous D2HG, in the presence or absence of the selective NMDA antagonist, AP5. Results: Preoperative seizures were observed in 18%–34% of IDH1 wild-type (IDH1wt) patients and in 59%–74% of IDH1mut patients (p < 0.001). Multivariable analysis, including WHO grade, 1p/19q codeletion, and temporal lobe location, showed that IDH1mut was an independent correlate with seizures (odds ratio 2.5, 95% confidence interval 1.6–3.9, p < 0.001). Exogenous D2HG increased the firing rate of cultured rat cortical neurons 4- to 6-fold, but was completely blocked by AP5. Conclusions: The D2HG product of IDH1mut may increase neuronal activity by mimicking the activity of glutamate on the NMDA receptor, and IDH1mut gliomas are more likely to cause seizures in patients. This has rapid translational implications for the personalized management of tumor-associated epilepsy, as targeted IDH1mut inhibitors may improve antiepileptic therapy in patients with IDH1mut gliomas.

Multiplexed RNAi therapy against brain tumor-initiating cells via lipopolymeric nanoparticle infusion delays glioblastoma progression

Significance Glioblastoma is a deadly brain tumor with no cure. Brain tumor-initiating cells (BTICs) have been recognized as the key driver behind the unstoppable malignant growth, therapy resistance, and recurrence. BTICs are exceptionally difficult to target because of heterogeneous genetic and epigenetic aberrations that are challenging to reverse therapeutically using conventional pharmaceuticals or biologics. Here we report a lipopolymeric nanoparticle (LPNP) formulation that demonstrates a surprisingly high affinity for BTICs and the capacity to encapsulate multiple siRNAs for potent and targeted anti-BTIC therapy. We show that direct infusion of LPNP siRNAs to brain tumors effectively impedes tumor growth in mouse and provides encouraging survival benefits. This multiplexed nanomedicine platform carries strong potential for personalized anti-BTIC therapies. Brain tumor-initiating cells (BTICs) have been identified as key contributors to therapy resistance, recurrence, and progression of diffuse gliomas, particularly glioblastoma (GBM). BTICs are elusive therapeutic targets that reside across the blood–brain barrier, underscoring the urgent need to develop novel therapeutic strategies. Additionally, intratumoral heterogeneity and adaptations to therapeutic pressure by BTICs impede the discovery of effective anti-BTIC therapies and limit the efficacy of individual gene targeting. Recent discoveries in the genetic and epigenetic determinants of BTIC tumorigenesis offer novel opportunities for RNAi-mediated targeting of BTICs. Here we show that BTIC growth arrest in vitro and in vivo is accomplished via concurrent siRNA knockdown of four transcription factors (SOX2, OLIG2, SALL2, and POU3F2) that drive the proneural BTIC phenotype delivered by multiplexed siRNA encapsulation in the lipopolymeric nanoparticle 7C1. Importantly, we demonstrate that 7C1 nano-encapsulation of multiplexed RNAi is a viable BTIC-targeting strategy when delivered directly in vivo in an established mouse brain tumor. Therapeutic potential was most evident via a convection-enhanced delivery method, which shows significant extension of median survival in two patient-derived BTIC xenograft mouse models of GBM. Our study suggests that there is potential advantage in multiplexed targeting strategies for BTICs and establishes a flexible nonviral gene therapy platform with the capacity to channel multiplexed RNAi schemes to address the challenges posed by tumor heterogeneity.

Non-tumor cell IDO1 predominantly contributes to enzyme activity and response to CTLA-4/PD-L1 inhibition in mouse glioblastoma

Glioblastoma (GBM) is the most common malignant brain tumor in adults with a median survival of 14.6months. A contributing factor to GBM aggressiveness is the intratumoral expression of the potently immunosuppressive enzyme, indoleamine 2,3 dioxygenase 1 (IDO1). The enzymatic activity of IDO1 is associated with the conversion of tryptophan into downstream kynurenine (Kyn), which has previously been hypothesized to contribute toward the suppression of tumor immunity. Utilizing the syngeneic, immunocompetent, intracranial GL261 cell GBM model, we previously demonstrated that tumor cell, but not non-tumor cell IDO1, suppresses T cell-mediated brain tumor regression in mice. Paradoxically, we also showed that the survival advantage mediated by immune checkpoint blockade is abrogated by non-tumor cell IDO1 deficiency. Here, we have built on our past observations and confirm the maladaptive role of tumor cell IDO1 in a novel mouse GBM model. We also demonstrate that, non-tumor cells, rather than mouse GBM cells, are the dominant contributor to IDO1-mediated enzyme activity. Finally, we show the novel associations between maximally-effective immune-checkpoint blockade-mediated survival, non-tumor cell IDO1 and intra-GBM Kyn levels. These data suggest for the first time that, GBM cell-mediated immunosuppression is IDO1 enzyme independent, while the survival benefits of immune checkpoint blockade require non-tumor cell IDO1 enzyme activity. Given that current clinical inhibitors vary in their mechanism of action, in terms of targeting IDO1 enzyme activity versus enzyme-independent effects, this work suggests that choosing an appropriate IDO1 pharmacologic will maximize the effectiveness of future immune checkpoint blockade approaches.

Peripheral plasma vitamin D and non-HDL cholesterol reflect the severity of cerebral cavernous malformation disease

AIM To correlate cerebral cavernous malformations (CCMs) disease aggressiveness with peripheral blood biomarkers hypothesized mechanistically. PATIENTS & METHODS A prospective case-control study enrolled 43 CCM patients, where 25-(OH) vitamin D, HDL and non-HDL cholesterol, CRP plasma levels and leukocyte ROCK activity were correlated with parameters of disease aggressiveness reflecting chronic and acute domains. RESULTS Patients with one or more features of chronically aggressive disease (early age at symptom onset, two or more symptomatic bleeds, high lesion burden) had significantly lower 25-(OH) vitamin D and non-HDL cholesterol levels in comparison to patients without these features. CONCLUSION Validation of these biomarkers and their potential treatment modulation may influence the clinical care of patients with CCM disease.

Infiltrating T Cells Increase IDO1 Expression in Glioblastoma and Contribute to Decreased Patient Survival

Purpose: Indoleamine 2,3 dioxygenase 1 (IDO1) mediates potent immunosuppression in multiple preclinical models of cancer. However, the basis for elevated IDO1 expression in human cancer, including the most common primary malignant brain tumor in adults, glioblastoma (GBM), is poorly understood. The major objective of this study is to address this gap in our understanding of how IDO1 expression contributes to the biology of GBM, and whether its level of expression is a determinant of GBM patient outcome. Experimental Design: Patient-resected GBM, The Cancer Genome Atlas, human T-cell:GBM cocultures, as well as nu/nu, NOD-scid, and humanized (NSG-SGM3-BLT) mice-engrafted human GBM form the basis of our investigation. Results: In situ hybridization for IDO1 revealed transcript expression throughout patient-resected GBM, whereas immunohistochemical IDO1 positivity was highly variable. Multivariate statistical analysis revealed that higher levels of IDO1 transcript predict a poor patient prognosis (P = 0.0076). GBM IDO1 mRNA levels positively correlated with increased gene expression for markers of cytolytic and regulatory T cells, in addition to decreased patient survival. Humanized mice intracranially engrafted human GBM revealed an IFNγ-associated T-cell–mediated increase of intratumoral IDO1. Conclusions: Our data demonstrate that high intratumoral IDO1 mRNA levels correlate with a poor GBM patient prognosis. It also confirms the positive correlation between increased GBM IDO1 levels and human-infiltrating T cells. Collectively, this study suggests that future efforts aimed at increasing T-cell–mediated effects against GBM should consider combinatorial approaches that coinhibit potential T-cell–mediated IDO1 enhancement during therapy. Clin Cancer Res; 23(21); 6650–60. ©2017 AACR.

Vascular permeability and iron deposition biomarkers in longitudinal follow-up of cerebral cavernous malformations

OBJECTIVE Vascular permeability and iron leakage are central features of cerebral cavernous malformation (CCM) pathogenesis. The authors aimed to correlate prospective clinical behavior of CCM lesions with longitudinal changes in biomarkers of dynamic contrast-enhanced quantitative permeability (DCEQP) and quantitative susceptibility mapping (QSM) assessed by MRI. METHODS Forty-six patients with CCMs underwent 2 or more permeability and/or susceptibility studies in conjunction with baseline and follow-up imaging and clinical surveillance during a mean 12.05 months of follow-up (range 2.4-31.27 months). Based on clinical and imaging features, cases/lesions were classified as stable, unstable, or recovering. Associated and predictive changes in quantitative permeability and susceptibility were investigated. RESULTS Lesional mean permeability and QSM values were not significantly different in stable versus unstable lesions at baseline. Mean lesional permeability in unstable CCMs with lesional bleeding or growth increased significantly (+85.9% change; p = 0.005), while mean permeability in stable and recovering lesions did not significantly change. Mean lesional QSM values significantly increased in unstable lesions (+44.1% change; p = 0.01), decreased slightly with statistical significance in stable lesions (-3.2% change; p = 0.003), and did not significantly change in recovering lesions. Familial cases developing new lesions during the follow-up period showed a higher background brain permeability at baseline (p = 0.001), as well as higher regional permeability (p = 0.003) in the area that would later develop a new lesion as compared with the homologous contralateral brain region. CONCLUSIONS In vivo assessment of vascular permeability and iron deposition on MRI can serve as objective and quantifiable biomarkers of disease activity in CCMs. This may be applied in natural history studies and may help calibrate clinical trials. The 2 techniques are likely applicable in other disorders of vascular integrity and iron leakage such as aging, hemorrhagic microangiopathy, and traumatic brain injury.

B-Cell Depletion Reduces the Maturation of Cerebral Cavernous Malformations in Murine Models

Cerebral cavernous malformations (CCMs) are relatively common vascular malformations, characterized by increased Rho kinase (ROCK) activity, vascular hyper-permeability and the presence of blood degradation products including non-heme iron. Previous studies revealed robust inflammatory cell infiltration, selective synthesis of IgG, in situ antigen driven B-cell clonal expansion, and deposition of immune complexes and complement proteins within CCM lesions. We aimed to evaluate the impact of suppressing the immune response on the formation and maturation of CCM lesions, as well as lesional iron deposition and ROCK activity. Two murine models of heterozygous Ccm3 (Pdcd10), which spontaneously develop CCM lesions with severe and milder phenotypes, were either untreated or received anti-mouse BR3 to deplete B cells. Brains from anti-mouse BR3-treated mice exhibited significantly fewer mature CCM lesions and smaller lesions compared to untreated mice. B cell depletion halted the progression of lesions into mature stage 2 lesions but did not prevent their genesis. Non-heme iron deposition and ROCK activity was decreased in lesions of B cell depleted mice. This represents the first report of the therapeutic benefit of B-cell depletion in the development and progression of CCMs, and provides a proof of principle that B cells play a critical role in CCM lesion genesis and maturation. These findings add biologics to the list of potential therapeutic agents for CCM disease. Future studies would characterize the putative antigenic trigger and further define the mechanism of immune response in the lesions.