Development of a new therapeutic regime for the treatment of glioblastoma multiforme (GBM)

Glioblastoma multiforme is the most frequently occurring primary brain tumour and it carries a dismal prognosis. Despite surgical intervention and aggressive chemo-radiotherapy the median survival of patients is just under 15 months. Very little progress has been made with regards to improving patient survival therefore novel therapeutic interventions are required. Vaccination offers an attractive option for the therapeutic treatment of GBM, with activated T-cells previously being shown to access tumours located within the brain resulting in improved survival in pre-clinical murine models of GBM. Several vaccine platforms have been developed and tested in GBM patients, however many of these therapies fail during clinical trials and as of 2019 no immunotherapeutic treatments have been approved for use in the GBM setting. One major obstacle to overcome when using the immune system to treat GBMs is the highly immunosuppressive phenotype these cancers have. GBMs frequently express immunosuppressive checkpoints that prevent anti-tumour T-cell activity. Immune checkpoint blockade has recently gained approval for the treatment of malignant melanoma, lung cancer, head and neck cancer, lymphoma, bladder cancer and kidney cancer. The utilisation of immune checkpoint blockade as a monotherapy improves survival in small subsets of patients however it is not a completely curative treatment modality. Therefore it is of great interest to see if combining immune checkpoint blockade with vaccine therapy can boost anti-tumour immunity by stimulating T-cells and preventing the inhibitory signals from tumour cells that prevent T-cell killing. Examination of GBM tumour tissues and cell lines revealed that a large proportion of GBMs express the immunogenic antigens Tyrosinase-related protein-2 (TRP-2) and Wilms’ tumour 1 (WT-1). It was also revealed that these tumours expressed several immunosuppressive proteins with PD-L1, HLA E and HLA-G expression being observed in tissues and cell lines studied. It was also revealed that when GBM cell lines were treated with the immune stimulating cytokine IFNγ they up-regulated the immunosuppressive proteins PD-L1 and IDO. The ImmunoBody® DNA plasmid vaccination encodes an IgG antibody molecule that acts as a carrier protein for the peptide targets of interest, these peptides are engrafted into the complementarity determining regions of the antibody. This method of vaccination generates a strong-immune response via direct and cross-presentation. Pre-clinical testing using the humanised HHDII/DR1 C57BL/6 mouse model revealed that a HLA-A2 specific TRP-2 and a HLA-A2 specific WT-1 directed ImmunoBody® vaccine generated a strong peptide specific immune response. When both vaccinations were given simultaneously this strong TRP-2 and WT-1 directed immune response was equivalent to when each vaccine was given alone meaning that epitope dominance is not a factor when targeting these two antigens. Using the HHDII/DR1 humanised mouse the effects of this dual ImmunoBody® vaccination regime was tested both prophylactically and therapeutically. In these proof of concept experiments the B16 HHDII/DR1 Luc2 cell line was utilised. This cell line expresses both TRP-2 and WT-1 antigens and it has the chimeric HLA-A2 HHDII/DR1 MHC molecule meaning it presents HLA-A2 specific peptides. When the dual ImmunoBody® vaccination regime was used prophylactically it significantly improved the survival of mice intracranially implanted with B16 HHDII/DR1 Luc2 cells compared to sham vaccinated mice. In the therapeutic setting the addition of an anti-PD-1 blocking antibody to the dual vaccination regime improved survival of B16 HHDII/DR1 Luc1 tumour bearing mice when compared to dual vaccinated mice receiving PD-1 isotype control antibody and control sham vaccinated mice that received PD-1 isotype antibody. Analysis of tumour infiltrating lymphocyte populations revealed that dual vaccination increases CD8+ T-cell infiltrate into these intracranial tumours with these cells showing increased cell surface expression of the activation markers CD25 and CD69. The dipeptide carnosine was also used to treat GBM cells in vitro, this molecule has previously been shown to have anti-tumour activity. When carnosine was used to treat GBM cells it led to reduced mitochondrial metabolism and migration of these cells. These properties make carnosine an attractive adjunct to immunotherapy. Overall these results provide promise for the use of ImmunoBody® vaccination with immune checkpoint blockade for the treatment of GBM. Whilst immune cells can actively access tumours systemically administered checkpoint antibodies don't cross the blood-brain-barrier freely, therefore in order for these therapies to be further developed methods for improving brain penetrance of checkpoint inhibitors needs to be explored

Targeting cellular pathways in glioblastoma multiforme

Glioblastoma multiforme (GBM) is a debilitating disease that is associated with poor prognosis, short median patient survival and a very limited response to therapies. GBM has a very complex pathogenesis that involves mutations and alterations of several key cellular pathways that are involved in cell proliferation, survival, migration and angiogenesis. Therefore, efforts that are directed toward better understanding of GBM pathogenesis are essential to the development of efficient therapies that provide hope and extent patient survival. In this review, we outline the alterations commonly associated with GBM pathogenesis and summarize therapeutic strategies that are aimed at targeting aberrant cellular pathways in GBM

Immune Escape in Glioblastoma Multiforme and the Adaptation of Immunotherapies for Treatment

Glioblastoma multiforme (GBM) is the most frequently occurring primary brain tumor and has a very poor prognosis, with only around 5% of patients surviving for a period of 5 years or more after diagnosis. Despite aggressive multimodal therapy, consisting mostly of a combination of surgery, radiotherapy, and temozolomide chemotherapy, tumors nearly always recur close to the site of resection. For the past 15 years, very little progress has been made with regards to improving patient survival. Although immunotherapy represents an attractive therapy modality due to the promising pre-clinical results observed, many of these potential immunotherapeutic approaches fail during clinical trials, and to date no immunotherapeutic treatments for GBM have been approved. As for many other difficult to treat cancers, GBM combines a lack of immunogenicity with few mutations and a highly immunosuppressive tumor microenvironment (TME). Unfortunately, both tumor and immune cells have been shown to contribute towards this immunosuppressive phenotype. In addition, current therapeutics also exacerbate this immunosuppression which might explain the failure of immunotherapy-based clinical trials in the GBM setting. Understanding how these mechanisms interact with one another, as well as how one can increase the anti-tumor immune response by addressing local immunosuppression will lead to better clinical results for immune-based therapeutics. Improving therapeutic delivery across the blood brain barrier also presents a challenge for immunotherapy and future therapies will need to consider this. This review highlights the immunosuppressive mechanisms employed by GBM cancers and examines potential immunotherapeutic treatments that can overcome these significant immunosuppressive hurdles

PYK2 promotes HER2-positive breast cancer invasion

Background: Metformin, a biguanide, is one of the most commonly prescribed treatments for type 2 diabetes and has recently been recommended as a potential drug candidate for advanced cancer therapy. Although Metformin has antiproliferative and proapoptotic effects on breast cancer, the heterogenous nature of this disease affects the response to metformin leading to the activation of pro-invasive signalling pathways that are mediated by the focal adhesion kinase PYK2 in pure HER2 phenotype breast cancer. Methods: The effect of metformin on different breast cancer cell lines, representing the molecular heterogenicity of the disease was investigated using in vitro proliferation and apoptosis assays. The activation of PYK2 by metformin in pure HER2 phenotype (HER2+/ER−/PR-) cell lines was investigated by microarrays, quantitative real time PCR and immunoblotting. Cell migration and invasion PYK2-mediated and in response to metformin were determined by wound healing and invasion assays using HER2+/ER−/PR- PYK2 knockdown cell lines. Proteomic analyses were used to determine the role of PYK2 in HER2+/ER−/PR- proliferative, migratory and invasive cellular pathways and in response to metformin. The association between PYK2 expression and HER2+/ER−/PR- patients’ cancer-specific survival was investigated using bioinformatic analysis of PYK2 expression from patient gene expression profiles generated by the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) study. The effect of PYK2 and metformin on tumour initiation and invasion of HER2+/ER−/PR- breast cancer stem-like cells was performed using the in vitro stem cell proliferation and invasion assays. Results: Our study showed for the first time that pure HER2 breast cancer cells are more resistant to metformin treatment when compared with the other breast cancer phenotypes. This drug resistance was associated with the activation of PTK2B/PYK2, a well-known mediator of signalling pathways involved in cell proliferation, migration and invasion. The role of PYK2 in promoting invasion of metformin resistant HER2 breast cancer cells was confirmed through investigating the effect of PYK2 knockdown and metformin on cell invasion and by proteomic analysis of associated cellular pathways. We also reveal a correlation between high level of expression of PYK2 and reduced survival in pure HER2 breast cancer patients. Moreover, we also report a role of PYK2 in tumour initiation and invasion-mediated by pure HER2 breast cancer stem-like cells. This was further confirmed by demonstrating a correlation between reduced survival in pure HER2 breast cancer patients and expression of PYK2 and the stem cell marker CD44. Conclusions: We provide evidence of a PYK2-driven pro-invasive potential of metformin in pure HER2 cancer therapy and propose that metformin-based therapy should consider the molecular heterogeneity of breast cancer to prevent complications associated with cancer chemoresistance, invasion and recurrence in treated patients

Consistent patterns of common species across tropical tree communities

Trees structure the Earth’s most biodiverse ecosystem, tropical forests. The vast number of tree species presents a formidable challenge to understanding these forests, including their response to environmental change, as very little is known about most tropical tree species. A focus on the common species may circumvent this challenge. Here we investigate abundance patterns of common tree species using inventory data on 1,003,805 trees with trunk diameters of at least 10 cm across 1,568 locations 1–6 in closed-canopy, structurally intact old-growth tropical forests in Africa, Amazonia and Southeast Asia. We estimate that 2.2%, 2.2% and 2.3% of species comprise 50% of the tropical trees in these regions, respectively. Extrapolating across all closed-canopy tropical forests, we estimate that just 1,053 species comprise half of Earth’s 800 billion tropical trees with trunk diameters of at least 10 cm. Despite differing biogeographic, climatic and anthropogenic histories 7, we find notably consistent patterns of common species and species abundance distributions across the continents. This suggests that fundamental mechanisms of tree community assembly may apply to all tropical forests. Resampling analyses show that the most common species are likely to belong to a manageable list of known species, enabling targeted efforts to understand their ecology. Although they do not detract from the importance of rare species, our results open new opportunities to understand the world’s most diverse forests, including modelling their response to environmental change, by focusing on the common species that constitute the majority of their trees

Anticancer actions of carnosine in cellular models of prostate cancer

Treatments for organ-confined prostate cancer include external beam radiation therapy, radical prostatectomy, radiotherapy/brachytherapy, cryoablation and high-intensity focused ultrasound. None of these are cancer-specific and are commonly accompanied by side effects, including urinary incontinence and erectile dysfunction. Moreover, subsequent surgical treatments following biochemical recurrence after these interventions are either limited or affected by the scarring present in the surrounding tissue. Carnosine (β-alanyl-L-histidine) is a histidine-containing naturally occurring dipeptide which has been shown to have an anti-tumorigenic role without any detrimental effect on healthy cells; however, its effect on prostate cancer cells has never been investigated. In this study, we investigated the effect of carnosine on cell proliferation and metabolism in both a primary cultured androgen-resistant human prostate cancer cell line, PC346Flu1 and murine TRAMP-C1 cells. Our results show that carnosine has a significant dose-dependent inhibitory effect in vitro on the proliferation of both human (PC346Flu1) and murine (TRAMP-C1) prostate cancer cells, which was confirmed in 3D-models of the same cells. Carnosine was also shown to decrease adenosine triphosphate content and reactive species which might have been caused in part by the increase in SIRT3 also shown after carnosine treatment. These encouraging results support the need for further human in vivo work to determine the potential use of carnosine, either alone or, most likely, as an adjunct therapy to surgical or other conventional treatments

Deep sequencing and automated histochemistry of human tissue slice cultures improve their usability as preclinical model for cancer research

Cancer research requires models closely resembling the tumor in the patient. Human tissue cultures can overcome interspecies limitations of animal models or the loss of tissue architecture in in vitro models. However, analysis of tissue slices is often limited to histology. Here, we demonstrate that slices are also suitable for whole transcriptome sequencing and present a method for automated histochemistry of whole slices. Tumor and peritumoral tissue from a patient with glioblastoma was processed to slice cultures, which were treated with standard therapy including temozolomide and X-irradiation. Then, RNA sequencing and automated histochemistry were performed. RNA sequencing was successfully accomplished with a sequencing depth of 243 to 368 x 106 reads per sample. Comparing tumor and peritumoral tissue, we identified 1888 genes significantly downregulated and 2382 genes upregulated in tumor. Treatment significantly downregulated 2017 genes, whereas 1399 genes were upregulated. Pathway analysis revealed changes in the expression profile of treated glioblastoma tissue pointing towards downregulated proliferation. This was confirmed by automated analysis of whole tissue slices stained for Ki67. In conclusion, we demonstrate that RNA sequencing of tissue slices is possible and that histochemical analysis of whole tissue slices can be automated which increases the usability of this preclinical model

Effects of probiotics and paraprobiotics on subjective and objective sleep metrics: A systematic review and meta-analysis

Khalesi, S ORCiD: 0000-0002-8208-2518Inadequate sleep (i.e., duration and/or quality) is becoming increasingly recognized as a global public health issue. Interaction via the gut-brain axis suggests that modification of the gut microbial environment via supplementation with live microorganisms (probiotics) or nonviable microorganisms/microbial cell fractions (paraprobiotics) may improve sleep health. This systematic review and meta-analysis aimed to clarify the effect of consuming probiotics/paraprobiotics on subjective and objective sleep metrics. Online databases were searched from 1980 to October 2019 for studies involving adults who consumed probiotics or paraprobiotics in controlled trials, during which, changes in subjective and/or objective sleep parameters were examined. A total of 14 studies (20 trials) were included in meta-analysis. Random effects meta-analyses indicated that probiotics/paraprobiotics supplementation significantly reduced Pittsburgh Sleep Quality Index (PSQI) score (i.e., improved sleep quality) relative to baseline (−0.78-points, 95% confidence interval: 0.395–1.166; p < 0.001). No significant effect was found for changes on other subjective sleep scales, nor objective parameters of sleep (efficiency/latency) measured using polysomnography or actigraphy. Subgroup analysis for PSQI data suggested that the magnitude of the effect was greater (although not statistically) in healthy participants than those with a medical condition, when treatment contained a single (rather than multiple) strain of probiotic bacteria, and when the duration of treatment was ≥8 weeks. Probiotics/paraprobiotics supplementation may have some efficacy in improving perceived sleep health, measured using the PSQI. While current evidence does not support a benefit of consuming probiotics/paraprobiotics when measured by other subjective sleep scales, nor objective measures of sleep; more studies using well-controlled, within-subject experimental designs are needed. © 2020, The Author(s), under exclusive licence to Springer Nature Limited