Adam M. Sonabend

Mesenchymal Stem Cells Effectively Deliver an Oncolytic Adenovirus to Intracranial Glioma

Gene therapy represents a promising treatment alternative for patients with malignant gliomas. Nevertheless, in the setting of these highly infiltrative tumors, transgene delivery remains a challenge. Indeed, viral vehicles tested in clinical trials often target only those tumor cells that are adjacent to the injection site. In this study, we examined the feasibility of using human mesenchymal stem cells (hMSC) to deliver a replication‐competent oncolytic adenovirus (CRAd) in a model of intracranial malignant glioma. To do so, CRAds with a chimeric 5/3 fiber or RGD backbone with or without CXCR4 promoter driving E1A were examined with respect to replication and toxicity in hMSC, human astrocytes, and the human glioma cell line U87MG by quantitative polymerase chain reaction and membrane integrity assay. CRAd delivery by virus‐loaded hMSC was then evaluated in vitro and in an in vivo model of mice bearing intracranial U87MG xenografts. Our results show that hMSC are effectively infected by CRAds that use the CXCR4 promoter. CRAd‐CXCR4‐RGD had the highest replication, followed by CRAd‐CXCR4–5/3, in hMSC, with comparable levels of toxicity. In U87MG tumor cells, CRAd‐CXCR4–5/3 showed the highest replication and toxicity. Virus‐loaded hMSC effectively migrated in vitro and released CRAds that infected U87MG glioma cells. When injected away from the tumor site in vivo, hMSC migrated to the tumor and delivered 46‐fold more viral copies than injection of CRAd‐CXCR4–5/3 alone. Taken together, these results indicate that hMSC migrate and deliver CRAd to distant glioma cells. This delivery strategy should be explored further, as it could improve the outcome of oncolytic virotherapy for glioma.

Stimulation of TLR9 with CpG ODN enhances apoptosis of glioma and prolongs the survival of mice with experimental brain tumors.

Toll‐like receptors (TLRs) recognize a set of conserved molecular structures, so called pathogen‐associated molecular patterns, which allow them to sense and initiate innate and adaptive immune responses. In this study, we examined the expression of TLRs in both human and murine glioma. We then analyzed the change in TLR expression after treatment with synthetic phosphorothioate oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides (CpG ODNs), strong activators of both innate and adaptive immunity. In addition, we investigated the in vivo effect of CpG injection into C57BL/6 mice implanted with syngeneic GL261 glioma. Our results indicate that TLR9 is overexpressed in human and murine glioma cell lines and CpG stimulation prolongs the survival of mice with experimental brain tumors. CpGs induce TLR9 down‐regulation, followed by apoptosis of GL261 cells in vitro as well as in vivo. Furthermore, the effects of CpG stimulation appear to enhance the antigen presenting capacity of microglia, shift the immune response toward CD8+ T cells, and decrease the number of CD4+CD25+ regulatory T cells. Taken together, our data support the role of CpG in glioma immunotherapy and provide a rationale for further clinical development of CpG therapy in patients with malignant glioma.

Inhibition of Sonic hedgehog and Notch pathways enhances sensitivity of CD133(+) glioma stem cells to temozolomide therapy.

Malignant gliomas are currently treated with temozolomide (TMZ), but often exhibit resistance to this agent. CD133+ cancer stem cells, a population believed to contribute to the tumor’s chemoresistance, bear the activation of Notch and Sonic hedgehog (SHH) pathways. In this study, we examined whether inhibition of both pathways enhances the efficacy of TMZ monotherapy in the context of glioma stem cells. Transcriptional analysis of Notch and SHH pathways in CD133+-enriched glioma cell populations showed the activity of these pathways. CD133+ cells were less susceptible to TMZ treatment than the unsorted glioma counterparts. Interestingly, Notch and SHH pathway transcriptional activity in CD133+ glioma cells was further enhanced by TMZ exposure, which led to NOTCH 1, NCOR2, and GLI1 upregulation (6.64-, 3.73-, and 2.79-fold, respectively) and CFLAR downregulation (4.22-fold). The therapeutic effect of TMZ was enhanced by Notch and SHH pathway pharmacological antagonism with GSI-1 and cyclopamine. More importantly, simultaneous treatment involving TMZ with both of these compounds led to a significant increase in CD133+ glioma cytotoxicity than treatment with any of these agents alone (P < 0.05). In conclusion, CD133+ glioma cells overexpress genes involved in Notch and SHH pathways. These pathways contribute to the chemoresistant phenotype of CD133+ glioma cells, as their antagonism leads to an additive effect when used in combination with TMZ.

Glioblastoma Models Reveal the Connection between Adult Glial Progenitors and the Proneural Phenotype

Background Tumor heterogeneity is a major obstacle for finding effective treatment of Glioblastoma (GBM). Based on global expression analysis, GBM can be classified into distinct subtypes: Proneural, Neural, Classical and Mesenchymal. The signatures of these different tumor subtypes may reflect the phenotypes of cells giving rise to them. However, the experimental evidence connecting any specific subtype of GBM to particular cells of origin is lacking. In addition, it is unclear how different genetic alterations interact with cells of origin in determining tumor heterogeneity. This issue cannot be addressed by studying end-stage human tumors. Methodology/Principal Findings To address this issue, we used retroviruses to deliver transforming genetic lesions to glial progenitors in adult mouse brain. We compared the resulting tumors to human GBM. We found that different initiating genetic lesions gave rise to tumors with different growth rates. However all mouse tumors closely resembled the human Proneural GBM. Comparative analysis of these mouse tumors allowed us to identify a set of genes whose expression in humans with Proneural GBM correlates with survival. Conclusions/Significance This study offers insights into the relationship between adult glial progenitors and Proneural GBM, and allows us to identify molecular alterations that lead to more aggressive tumor growth. In addition, we present a new preclinical model that can be used to test treatments directed at a specific type of GBM in future studies.

Neural stem cells target intracranial glioma to deliver an oncolytic adenovirus in vivo

Adenoviral oncolytic virotherapy represents an attractive treatment modality for central nervous system (CNS) neoplasms. However, successful application of virotherapy in clinical trials has been hampered by inadequate distribution of oncolytic vectors. Neural stem cells (NSCs) have been shown as suitable vehicles for gene delivery because they track tumor foci. In this study, we evaluated the capability of NSCs to deliver a conditionally replicating adenovirus (CRAd) to glioma. We examined NSC specificity with respect to viral transduction, migration and capacity to deliver a CRAd to tumor cells. Fluorescence-activated cell sorter (FACS) analysis of NSC shows that these cells express a variety of surface receptors that make them amenable to entry by recombinant adenoviruses. Luciferase assays with replication-deficient vectors possessing a variety of transductional modifications targeted to these receptors confirm these results. Real-time PCR analysis of the replication profiles of different CRAds in NSCs and a representative glioma cell line, U87MG, identified the CRAd-Survivin (S)-pk7 virus as optimal vector for further delivery studies. Using in vitro and in vivo migration studies, we show that NSCs infected with CRAd-S-pk7 virus migrate and preferentially deliver CRAd to U87MG glioma. These results suggest that NSCs mediate an enhanced intratumoral distribution of an oncolytic vector in malignant glioma when compared with virus injection alone.

MRI-localized biopsies reveal subtype-specific differences in molecular and cellular composition at the margins of glioblastoma.

Significance Molecular analysis of surgically resected glioblastomas (GBM) samples has uncovered phenotypically and clinically distinct tumor subtypes. However, little is known about the molecular features of the glioma margins that are left behind after surgery. To address this key issue, we performed RNA-sequencing (RNA-seq) and histological analysis on MRI-guided biopsies from the contrast-enhancing core and nonenhancing margins of GBM. Computational deconvolution of the RNA-seq data revealed that cellular composition, including nonneoplastic cells, is a major determinant of the expression patterns at the margins of GBM. The different GBM subtypes show distinct expression patterns that relate the contrast enhancing centers to the nonenhancing margins of tumors. Understanding these patterns may provide a means to infer the molecular and cellular features of residual disease. Glioblastomas (GBMs) diffusely infiltrate the brain, making complete removal by surgical resection impossible. The mixture of neoplastic and nonneoplastic cells that remain after surgery form the biological context for adjuvant therapeutic intervention and recurrence. We performed RNA-sequencing (RNA-seq) and histological analysis on radiographically guided biopsies taken from different regions of GBM and showed that the tissue contained within the contrast-enhancing (CE) core of tumors have different cellular and molecular compositions compared with tissue from the nonenhancing (NE) margins of tumors. Comparisons with the The Cancer Genome Atlas dataset showed that the samples from CE regions resembled the proneural, classical, or mesenchymal subtypes of GBM, whereas the samples from the NE regions predominantly resembled the neural subtype. Computational deconvolution of the RNA-seq data revealed that contributions from nonneoplastic brain cells significantly influence the expression pattern in the NE samples. Gene ontology analysis showed that the cell type-specific expression patterns were functionally distinct and highly enriched in genes associated with the corresponding cell phenotypes. Comparing the RNA-seq data from the GBM samples to that of nonneoplastic brain revealed that the differentially expressed genes are distributed across multiple cell types. Notably, the patterns of cell type-specific alterations varied between the different GBM subtypes: the NE regions of proneural tumors were enriched in oligodendrocyte progenitor genes, whereas the NE regions of mesenchymal GBM were enriched in astrocytic and microglial genes. These subtype-specific patterns provide new insights into molecular and cellular composition of the infiltrative margins of GBM.

Prevention of ventriculostomy-related infections with prophylactic antibiotics and antibiotic-coated external ventricular drains: a systematic review.

BACKGROUND:Ventriculostomy-related infection (VRI) is a severe complication of external ventricular drain use, occurring in 5% to 23% of patients. Preventive measures for VRI include prolonged prophylactic systemic antibiotics (PSAs) and an antibiotic-coated external ventricular drains (ac-EVDs). OBJECTIVE:We performed a systematic review of all studies evaluating PSAs and ac-EVD for VRI prevention through July 2010. METHODS:Two reviewers independently assessed eligibility and evaluated study quality based on pre-established criteria. Observational studies and randomized clinical trials (RCTs) that fulfilled inclusion criteria were included in the meta-analysis. RESULTS:Three RCTs and 7 observational studies met our inclusion criteria and were included in the analysis. The type of antibiotics and VRI definitions varied among these studies. Pooled analysis showed a protective effect of PSAs and ac-EVDs for VRI (risk ratio: 0.32; 95% CI: 0.18-0.56). Results showed moderate heterogeneity (I2 = 53%) explained by the difference in quality among the studies and the inclusion of 1 large positive cohort study. The effect of PSAs and ac-EVDs was unrelated to the type of study (RCT or observational, P for interaction = .55), the route of antibiotic administration (PSAs or ac-EVDs, P = .13), or the quality of the studies (suboptimal vs good/excellent, P = .55). CONCLUSION:RCTs and observational-derived evidence support the use of PSAs throughout the duration of external ventricular drainage; similarly, the use of ac-EVDs to prevent VRI seems to be beneficial. Available data are heterogeneous and of suboptimal quality. Further research is needed to confirm the findings of this meta-analysis. There are not sufficient data to compare the protective effect of ac-EVDs and PSAs.

Combination of adenoviral virotherapy and temozolomide chemotherapy eradicates malignant glioma through autophagic and apoptotic cell death in vivo

Conditionally replicative adenoviruses (CRAds) represent a novel treatment strategy for malignant glioma. Recent studies suggest that the cytopathic effect elicited by these vectors is mediated through autophagy, a form of programmed cell death. Likewise, temozolomide (TMZ), a chemotherapeutic agent used for the treatment of malignant gliomas, also triggers autophagic cell death. In this study, we examined the potential to combine the two treatments in the setting of experimental glioma. In vitro, pretreatment with TMZ followed by CRAd-Surivin-pk7 enhanced cytotoxicity against a panel of glioma cell lines. Western blot analysis showed increased expression of BAX and p53, decreased expression of BCL2 and elevated level of APG5. Treatment with TMZ followed by CRAd-Survivin-pk7 (CRAd-S-pk7) led to a significant over-expression of autophagy markers, acidic vesicular organelles and light-chain 3 (LC3). These results were further evaluated in vivo, in which 90% of the mice with intracranial tumours were long-term survivors (>100 days) after treatment with TMZ and CRAd-S-pk7 (P<0.01). Analysis of tumours ex vivo showed expression of both LC3 and cleaved Caspase-3, proving that both autophagy and apoptosis are responsible for cell death in vivo. These results suggest that combination of chemovirotherapy offers a powerful tool against malignant glioma and should be further explored in the clinical setting.

Comparative evaluation of survivin, midkine, and CXCR4 promoters for transcriptional targeting of glioma gene therapy

Objective: Transcriptional targeting is a key strategy to enhance therapeutic efficacy of gene therapy applications. In the context of oncolytic virotherapy, transcriptional promoter elements are used from genes that are over expressed in a variety of malignant cancers. In the present study, we examined the feasibility of transcriptional targeting to glioma cells by comparing the activity of survivin, midkine, and CXCR4 tumor-specific promoters. Methods: To evaluate the expression level of several glioma related genes, we performed quantitative PCR analyses on samples obtained from cell lines and patients. To determine specific level of gene expression mediated by selective promoter elements, we measured luciferase expression in glioma samples transduced with replication deficient adenoviral vectors. Finally, we incorporated the optimal promoters into a conditionally replicative adenoviral vector, CRAd-5/3, and examined the cytopathic effect in vitro. Results: The survivin promoter demonstrated the highest level of mRNA expression in primary tumor samples and cell lines. Transcriptional targeting was confirmed by infection of glioma cells with an adenovirus expression vector containing a survivin-driven luciferase reporter gene. Of the tested promoters, minimal level of survivin activity was detected in normal human liver and brain. A novel vector, CRAd-5/3, with E1a under the control of the survivin promoter, exhibited enhanced cytopathic effect in vitro. Conclusions: Our data demonstrate that the survivin promoter element is very active in glioma samples and has low activity in normal human brain and liver. A novel oncolytic virus, CRAd-survivin-5/3, was effective against a panel of glioma cell lines in vitro. Our results suggest that employing the survivin promoter element in the context of CRAd-5/3 may present a new opportunity for the development of glioma specific oncolytic vectors.

Enhanced transduction of malignant glioma with a double targeted Ad5/3-RGD fiber-modified adenovirus

Malignant brain tumors remain refractory to adenovirus type 5 (Ad5)–based gene therapy, mostly due to the lack of the primary Ad5 receptor, the coxsackie and adenovirus receptor, on brain tumor cells. To bypass the dependence on coxsackie and adenovirus receptor for adenoviral entry and infectivity, we used a novel, double targeted Ad5 backbone–based vector carrying a chimeric Ad5/3 fiber with integrin-binding RGD motif incorporated in its Ad3 knob domain. We then tested the new virus in vitro and in vivo in the setting of malignant glioma. Ad5/3-RGD showed a 10-fold increase in gene expression in passaged cell lines and up to 75-fold increase in primary tumors obtained from patients relative to the control. These results were further corroborated in our in vivo human glioma xenograft model, where the Ad5/3-RGD vector showed a 1,000-fold increase in infectivity as compared with the control. Taken together, our findings indicate that Ad5/3-RGD may be a superior vector for applications in glioma gene therapy and therefore warrants further attention in the field of neuro-oncology. [Mol Cancer Ther 2006;5(9):2408–17]