The oncolytic virus Delta-24-RGD elicits an antitumor effect in pediatric glioma and DIPG mouse models

Pediatric high-grade glioma (pHGG) and diffuse intrinsic pontine gliomas (DIPGs) are aggressive pediatric brain tumors in desperate need of a curative treatment. Oncolytic virotherapy is emerging as a solid therapeutic approach. Delta-24-RGD is a replication competent adenovirus engineered to replicate in tumor cells with an aberrant RB pathway. This virus has proven to be safe and effective in adult gliomas. Here we report that the administration of Delta-24-RGD is safe in mice and results in a significant increase in survival in immunodeficient and immunocompetent models of pHGG and DIPGs. Our results show that the Delta-24-RGD antiglioma effect is mediated by the oncolytic effect and the immune response elicited against the tumor. Altogether, our data highlight the potential of this virus as treatment for patients with these tumors. Of clinical significance, these data have led to the start of a phase I/II clinical trial at our institution for newly diagnosed DIPG (NCT03178032)

Immuno-virotherapy for glioblastoma: Characterization of Delta 24-ACT in combination with different immunomodulators as therapeutic approach

The tumor microenvironment of glioblastoma is extremely immunosuppressive due to high amount of myeloid cells leading to low levels of lymphocytes. This immunosuppressive scenario makes difficult the development of curative therapies for this devastating tumor. Our group has engineered Delta-24-ACT, an oncolytic adenovirus armed with the costimulatory ligand 4-1BBL which is capable to trigger the activation of T cells and thereby, increasing the antitumor response. Since, monotherapies have not elicited survival benefit in glioblastoma, in this project; we evaluated the antitumor effect of Delta-24-ACT combined with different modulators of the immune system (targeting both lymphoid and myeloid cell populations). Delta-24-ACT was able to infect and kill murine (GL261-5 and CT-2A) and human (U87-MG and U251-MG) glioma cell lines, while maintaining its replication in the latter. Of importance, after infection with Delta-24-ACT, the 4-1BBL not only was detected on the membrane of glioma cells but also it was able to stimulate CD8 T cells in vitro. These data suggested its potential to trigger an effective immune response. Furthermore, in vivo, Delta-24-ACT significantly increased the median survival and led to long-term survivors in three different orthotopic glioma models. However, the virus did not generate antiglioma memory immune response. In order to improve the survival and to generate immune memory, we combined the virus with different immune modulating approaches (dendritic cell vaccination, IDO inhibitor, macrophages inhibitor etc). However, no differences in survival benefit were observed when compared to Delta-24-ACT. Since Delta-24-ACT treatment led to the expression of higher levels of PD-1 in T cells, we combined Delta-24-ACT with an anti-PD-L1 antibody. This combination not only alleviated the exhaustion phenotype showed by the T-cells but in addition, resulted in an increase survival and the acquisition of antiglioma memory immune response. In summary, our data demonstrated that Delta-24-ACT exerts a potent antitumor response in vitro and in vivo. Moreover, the different proposed combination strategies induce survival benefit in mice as a result of the recruitment of immune cell populations modulating the immunosuppressive tumor microenvironment of gliomas. Of importance, Delta-24-ACT in combination with PD-L1 blockade displayed the best therapeutic effect

Transcriptome Analysis of Ullrich Congenital Muscular Dystrophy Fibroblasts Reveals a Disease Extracellular Matrix Signature and Key Molecular Regulators

Collagen VI related myopathies encompass a range of phenotypes with involvement of skeletal muscle, skin and other connective tissues. They represent a severe and relatively common form of congenital disease for which there is no treatment. Collagen VI in skeletal muscle and skin is produced by fibroblasts. In order to gain insight into the consequences of collagen VI mutations and identify key disease pathways we performed global gene expression analysis of dermal fibroblasts from patients with Ullrich Congenital Muscular Dystrophy with and without vitamin C treatment. The expression data were integrated using a range of systems biology tools. Results were validated by real-time PCR, western blotting and functional assays. We found significant changes in the expression levels of almost 600 genes between collagen VI deficient and control fibroblasts. Highly regulated genes included extracellular matrix components and surface receptors, including integrins, indicating a shift in the interaction between the cell and its environment. This was accompanied by a significant increase in fibroblasts adhesion to laminin. The observed changes in gene expression profiling may be under the control of two miRNAs, miR-30c and miR-181a, which we found elevated in tissue and serum from patients and which could represent novel biomarkers for muscular dystrophy. Finally, the response to vitamin C of collagen VI mutated fibroblasts significantly differed from healthy fibroblasts. Vitamin C treatment was able to revert the expression of some key genes to levels found in control cells raising the possibility of a beneficial effect of vitamin C as a modulator of some of the pathological aspects of collagen VI related diseases

Integrin-α3 expression in collagen VI deficient fibroblasts.

<p>A. Immunofluorescence for integrin-α3 in confluent fibroblast cultures. B. Representative western blot analysis. The intensity of the bands corresponding to integrin-α3 (ITGA3) was quantified by densitometry using α-tubulin (A-TUB) as a loading control and expressed as arbitrary units relative to the control samples.</p

Cell adhesion assay.

<p>Adherence of UCMD fibroblasts (n = 7) relative to control fibroblasts (n = 7) for fibronectin, vitronectin, laminin and collagen type I (experiments were performed in triplicate). To normalize adhesion values between experiments, we expressed the results as a ratio between the absorbance values for collagen type IV (which was the substrate that showed the smallest variability between individual cultures and experiments, data not shown) and each ECM protein, (student t-test * p < 0.05).</p

Co-variance analysis was used to plot the expression values of a selection of genes of interest in untreated patient cells (P), control cells (C) and patient cells treated with AA (P_AA).

<p>Median and ranges are represented. A. HLA-DRA, B. COL1A1, C. TNC, D. TNX.</p

Ingenuity Pathway Analysis.

<p>Graphic representation of the network “cell cycle, skeletal and muscular system development”. Nodes represent genes and lines show the relationship between genes. The intensity of the node color indicates the degree of the up-regulation (red) or down-regulation (green) of significant genes in the P-C comparison. Non-color nodes are added by the tool. For a detailed legend refer to <a href="http://ingenuity.force.com/ipa/articles/Feature_Description/Legend" target="_blank">http://ingenuity.force.com/ipa/articles/Feature_Description/Legend</a>.</p

Extracellular matrix gene correlation network.

<p>This network represents Pearson correlations (R >0.8, p-value<0.005) computed considering expression values of genes of interest according to the microarray data and using Cytoscape tool. We selected those genes on the ECM-receptor interaction KEGG pathway and COL6A genes. Continuous lines represent positive correlations and discontinuous lines negative ones. Those correlations that are significant in patients´cells only are represented in black lines. Orange lines represent those correlations that are present in both patients and control cells but are of different sign (positive or negative) whereas those that have the same sign are represented by lilac lines. Red lines around gene symbols represent significantly over-expressed genes and green lines those that were under-expressed in patients´ fibroblasts relative to control fibroblasts.</p

miRNAs expression analysis.

<p>Real-time PCR was used to measure relative expression of miR-181a and miR-30c in skeletal muscle (A) and fibroblasts (B) from UCMD patients and in serum samples from UCMD, BM and DMD patients for miR-181a (C) and miR-30c (D). miRNA expression level was normalized against U6 miRNA. Results were calculated relative to control samples and are represented as mean and standard error.</p

Monocyte depletion enhances neutrophil influx and proneural to mesenchymal transition in glioblastoma

Myeloid cells are the predominant cell type in the tumor microenvironment of human and murine glioblastoma (GBM). By generating a mouse model deficient for all monocyte chemoattractant proteins, here the authors show that blocking monocyte recruitment promotes a compensatory neutrophil influx and that concomitant neutrophil inhibition is required to improve survival in GBM preclinical models