Development of an anti-invasive therapeutic strategy for paediatric high grade glioma

Paediatric high grade glioma (pHGG) and diffuse intrinsic pontine glioma (DIPG) are highly aggressive brain tumours. Their invasive phenotype contributes to their limited therapeutic response and novel treatments that block brain tumour invasion are needed. Here, two novel anti-invasive therapeutic strategies are evaluated: glycogen synthase kinase-3 (GSK-3) inhibitors and oncolytic viruses (OVs). Small molecule GSK-3 inhibitors, lithium chloride (LiCl) and the indirubin derivative 6-bromoindirubin-oxime (BIO), reduced migration and invasion of pHGG cell lines (SF188 and KNS42) and one patient-derived DIPG cell line (HSJD-DIPG-007) in 2D and 3D in vitro assays. Following drug treatment, pHGG cells demonstrated loss of polarity and altered morphology as seen by live cell imaging and cytoskeletal rearrangement of actin fibres and focal adhesions as seen by immunofluorescence. OVs (herpes simplex virus (HSV), reovirus and vaccinia virus (VV)) were able to inhibit the migration and invasion of pHGG and DIPG cell lines. Oncolytic HSV was the most interesting candidate, as anti-migratory and anti-invasive effects did not appear to be a consequence of cytotoxicity or altered proliferation. Oncolytic HSV altered pHGG cytoskeletal dynamics, stabilising microtubules through the accumulation of post-translational tubulin modifications, as evaluated by Western blotting and immunofluorescent labelling. Furthermore, oncolytic HSV treatment of pHGG cell lines inhibited GSK-3β activity and prevented the localised clustering of adenomatous polyposis coli to the leading edge of the cell. These observations are highly novel and begin to document the molecular mechanisms by which oncolytic HSV may inhibit pHGG migration and invasion. In conclusion, this study is the first to demonstrate that it is possible to target migration and invasion of pHGG and DIPG in vitro using either small molecule GSK-3 inhibitors or OVs, such as HSV. These agents warrant further in vivo pre-clinical investigation as potential anti-invasive therapeutics and have the potential to improve outcomes of these devastating childhood diseases

High-content analysis of tumour cell invasion in three-dimensional spheroid assays

Targeting infiltrating tumour cells is an attractive way of combating cancer invasion and metastasis. Here we describe a novel and reproducible method for high content analysis of invading cells using multicellular tumour spheroid assays in a high grade glioma model. Live cell imaging of spheroids generated from glioma cell lines, U87 and U251, gave insight into migration dynamics and cell morphology in response to anti-migratory drugs. Immunofluorescence imaging confirmed cytoskeletal rearrangements in the treated cells indicating a direct effect on cell morphology. Effect on migration was determined by a Migration Index (MI) from brightfield images which confirmed anti-migratory activity of the drugs. A marked effect on the core with treatment suggestive of disordered proliferation was also observed. A newly developed technique to prepare the spheroids and migratory cells for immunohistochemistry allowed an assessment of response to drug treatment with a selection of markers. A difference in protein expression was noted between cells maintained within the core and migratory cells indicative of the presence of cell subpopulations within the spheroid core. We conclude that this high content analysis allows researchers to perform screening of anti-tumour invasion compounds and study their effects on cellular dynamics, particularly in relation to protein expression, for the first time.</p

Critical review of current clinical practice guidelines for antifungal therapy in paediatric haematology and oncology

PURPOSE: The incidence of invasive fungal disease (IFD) is rising, but its treatment in paediatric haematology and oncology patients is not yet standardised. This review aimed to critically appraise and analyse the clinical practice guidelines (CPGs) that are available for paediatric IFD. METHODS: Electronic searches of MEDLINE, MEDLINE in-Process & Other non-Indexed Citations, the Guidelines International Network (GIN), guideline.gov and Google were performed and combined fungal disease (Fung* OR antifung*OR Candida* OR Aspergill*) with prophylaxis or treatment (prophyl* OR therap* OR treatment). All guidelines were assessed using the AGREE II tool and recommendations relating to prophylaxis, empirical treatment and specific therapy were extracted. RESULTS: Nineteen guidelines met the inclusion criteria. The AGREE II scores for the rigour of development domain ranged from 11 to 92 % with a median of 53 % (interquartile range 32-69 %). Fluconazole was recommended as antifungal prophylaxis in all nine of the included guidelines which recommended a specific drug. Liposomal amphotericin B was recommended in all five guidelines giving empirical therapy recommendations. Specific therapy recommendations were given for oral or genital candidiasis, invasive candida infection, invasive aspergillosis and other mould infections. CONCLUSIONS: In many areas, recommendations were clear about appropriate practice but further clarity was required, particularly relating to the decision to discontinue empirical antifungal treatment, the relative benefits of empiric and pre-emptive strategies and risk stratification. Future CPGs could consider working to published guideline production methodologies and sharing summaries of evidence appraisal to reduce duplication of effort, improving the quality and efficiency of CPGs in this area

Pediatric pan-central nervous system tumor analysis of immune-cell infiltration identifies correlates of antitumor immunity

Here, using methylCIBERSORT, the authors characterize the tumour-immune microenvironment of paediatric central nervous system (CNS) tumours and its association with tumour type and prognosis. These findings suggest that immuno-methylomic profiling may inform immunotherapy approaches in paediatric patients with CNS tumour

Combination viroimmunotherapy with checkpoint inhibition to treat glioma, based on location-specific tumor profiling

Background Systemic delivery of a complementary cDNA library expressed from the vesicular stomatitis virus (VSV) treats tumors by vaccinating against a wide range of tumor associated antigens (TAAs). For subcutaneous B16 melanomas, therapy was achieved using a specific combination of self-TAAs (neuroblastoma-Ras, cytochrome c, and tyrosinase-related protein 1) expressed from VSV. However, for intracranial B16 tumors, a different combination was therapeutic (consisting of VSV-expressed hypoxia-inducible factor [HIF]–2α, Sox-10, c-Myc, and tyrosinase-related protein 1). Therefore, we tested the hypothesis that tumors of different histological types growing in the brain share a common immunogenic signature which can be exploited for immunotherapy. Methods Syngeneic tumors, including GL261 gliomas, in the brains of immune competent mice were analyzed for their antigenic profiles or were treated with systemic viroimmunotherapy. Results Several different histological types of tumors growing intracranially, as well as freshly resected human brain tumor explants, expressed a HIF-2αHi phenotype imposed by brain-derived CD11b+ cells. This location-specific antigen expression was exploited therapeutically against intracranial GL261 gliomas using systemically delivered VSV expressing HIF-2α, Sox-10, and c-Myc. Viroimmunotherapy was enhanced by immune checkpoint inhibitors, associated with the de-repression of antitumor T-helper cell type 1 (Th1) interferon-γ and Th17 T cell responses. Conclusions Since different tumor types growing in the same location in the brain share a location-specific phenotype, we suggest that antigen-specific immunotherapies should be based upon expression of both histological type–specific tumor antigens and location-specific antigens. Our findings support clinical application of VSV-TAA therapy with checkpoint inhibition for aggressive brain tumors and highlight the importance of the intracranial microenvironment in sculpting a location-specific profile of tumor antigen expression