The effects of CHF6467, a new mutated form of NGF, on cell models of human glioblastoma. A comparison with wild-type NGF.

CHF6467 is a mutated form of human recombinant nerve growth factor (NGF). The mutation selectively disrupts the binding of NGF to its p75NTR receptor while maintaining the affinity toward TrkA receptor. Because of such different profile of receptor interaction, CHF6467 maintains unaltered the neurotrophic and neuroprotective properties of wild-type NGF but shows reduced algogenic activity.In this study, we investigated the effects of CHF6467 on mortality, proliferation, cell-damage and migration in three human glioblastoma cell lines (U87MG, T98G, LN18), and in the rat astrocytoma C6 cells. Both CHF6467 and wild-type NGF, given in the range 1-50 ng/ml, did not modify cell proliferation, metabolism and migration, as well as the number of live/dead cells.The present in vitro data are predictive of a lack of tumorigenic activity by both wild-type NGF and CHF6467 on these cell types in vivo, and warrant for CHF6467 further clinical development.

PDIA3 Expression in Glioblastoma Modulates Macrophage/Microglia Pro-Tumor Activation.

The glioblastoma (GB) microenvironment includes cells of the innate immune system identified as glioma-associated microglia/macrophages (GAMs) that are still poorly characterized. A potential role on the mechanisms regulating GAM activity might be played by the endoplasmic reticulum protein ERp57/PDIA3 (protein disulfide-isomerase A3), the modulation of which has been reported in a variety of cancers. Moreover, by using The Cancer Genome Atlas database, we found that overexpression of PDIA3 correlated with about 55% reduction of overall survival of glioma patients. Therefore, we analyzed the expression of ERp57/PDIA3 using specimens obtained after surgery from 18 GB patients. Immunohistochemical analysis of tumor samples revealed ERp57/PDIA3 expression in GB cells as well as in GAMs. The ERp57/PDIA3 levels were higher in GAMs than in the microglia present in the surrounding parenchyma. Therefore, we studied the role of PDIA3 modulation in microglia-glioma interaction, based on the ability of conditioned media collected from human GB cells to induce the activation of microglial cells. The results indicated that reduced PDIA3 expression/activity in GB cells significantly limited the microglia pro-tumor polarization towards the M2 phenotype and the production of pro-inflammatory factors. Our data support a role of PDIA3 expression in GB-mediated protumor activation of microglia.

The effects of painless nerve growth factor on human microglia polarization.

Previous studies in the rat suggest that microglial cells represent a potential druggable target for nerve growth factor (NGF) in the brain. The painless human Nerve Growth Factor (hNGFp) is a recombinant mutated form of human nerve growth factor (hNGF) that shows identical neurotrophic and neuroprotective properties of wild-type NGF but displays at least 10-fold lower algogenic activity. From the pharmacological point of view, hNGFp is a biased tropomyosin receptor kinase A (TrkA) agonist and displays a significantly lower affinity for the p75 neurotrophin receptor (p75NTR). This study aimed to evaluate the expression of TrkA and p75NTR NGF receptors in two different human microglia cell lines, and to investigate the effects of hNGFp and wild-type NGF (NGF) on L-arginine metabolism, taken as a marker of microglia polarization. Both NGF receptors are expressed in human microglia cell lines and are effective in transducing signals triggered by NGF and hNGFp. The latter and, to a lesser extent, NGF inhibit cytokine-stimulated inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in these cells. Conversely NGF but not hNGFp stimulates arginase-mediated urea production.

Vascular endothelial growth factor receptor 1 in glioblastoma­associated microglia/macrophages.

The anti­vascular endothelial growth factor­A (VEGF­A) monoclonal antibody (mAb) bevacizumab is an FDA­approved monotherapy for the treatment of recurrent glioblastoma (GB), a highly angiogenic and infiltrative tumour. However, bevacizumab does not increase overall survival and blockade of VEGF­A/VEGF receptor (VEGFR)­2 signal transduction is associated with severe adverse effects due to inhibition of physiological angiogenesis. Conversely, VEGFR­1 does not play a relevant role in physiological angiogenesis in the adult. VEGFR­1 is activated by both VEGF­A and placenta growth factor (PlGF), a protein involved in tumour growth and progression. In previous studies, it was demonstrated that inhibition of VEGFR­1 using a specific mAb developed in our laboratories reduced angiogenesis and GB cell chemotaxis and increased the survival of tumour­bearing mice. Failure of treatments directed toward the VEGF­A/VEGFR­2 axis could in part be due to inefficient targeting of the tumour microenvironment. In the present study, VEGFR­1 expression was investigated in GB­associated microglia/macrophages (GAMs) by analysing surgical specimens collected from 42 patients with GB. Data obtained from The Cancer Genome Atlas (TCGA) database revealed that upregulation of the VEGFR­1 ligands VEGF­A and PlGF was associated with a significant reduction in overall survival for patients with GB, highlighting the potential relevance of this receptor in the aggressiveness of GB. Immunohistochemical analysis indicated that VEGFR­1 is expressed not only in GB tissue but also in GAMs. Furthermore, the percentage of VEGFR­1­positive GAMs was significantly higher in the tumour region compared with that noted in the surrounding parenchyma. Thus, VEGFR­1 represents a potential therapeutic target for the treatment of GB, being present not only in GB and endothelial cells, but also in GAMs that are involved in tumour progression.

DNA inhibitors for the treatment of brain tumors.

Introduction: The worldwide incidence of central nervous system (CNS) primary tumors is increasing. Most of the chemotherapeutic agents used for treating these cancer types induce DNA damage, and their activity is affected by the functional status of repair systems involved in the detection or correction of DNA lesions. Unfortunately, treatment of malignant high-grade tumors is still an unmet medical need.Areas covered: We summarize the action mechanisms of the main DNA inhibitors used for the treatment of brain tumors. In addition, studies on new agents or drug combinations investigated for this indication are reviewed, focusing our attention on clinical trials that in the last 3 years have been completed, terminated or are still recruiting patients.Expert opinion: Much still needs to be done to render aggressive CNS tumors curable or at least to transform them from lethal to chronic diseases, as it is possible for other cancer types. Drugs with improved penetration in the CNS, toxicity profile, and activity against primary and recurrent tumors are eagerly needed. Targeted agents with innovative mechanisms of action and ability to harness the cells of the tumor microenvironment against cancer cells represent a promising approach for improving the clinical outcome of CNS tumors.

Pro-Inflammatory Activation of A New Immortalized Human Microglia Cell Line.

: The characterization of human microglia has been hampered by poor availability of human cell sources. However, microglia is involved in the physiopathology of multiple sclerosis, Alzheimer's disease, Parkinson's disease, HIV dementia, retinal degenerative diseases, cancer, and many other conditions. Therefore, there is an important need to have experimental paradigms of human microglia characterized and usable to study the role of microglia in the different pathologies in which it is involved. In the present work, we carried out an extensive characterization of Immortalized Human Microglia-SV40 cell line (IMhu), marketed by Applied Biological Material. The functional response of IMhu to a large variety of stimuli was studied. In particular, we investigated morphology, mortality, and changes in the production of different cytokines and chemokines, both under basal conditions and after stimulation. Moreover, western blotting analysis was conducted on phospho-mTOR (Ser 2448) and downstream parameters, p-P70S6K and 4EBP1, in order to understand if IMhu can be used for evaluations of mTOR pathway. In conclusion, IMhu cells proved to be a useful experimental model to investigate the physiopathology of inflammatory disease that involved microglia cells, including pathological conditions that involved the mTOR pathway.

Phospho-mTOR expression in human glioblastoma microglia-macrophage cells.

The glioblastoma (GBM) immune microenvironment is highly heterogeneous, and microglia may represent 30-70% of the entire tumor. However, the role of microglia and other specific immune populations is poorly characterized. Activation of mTOR signaling occurs in numerous human cancers and has roles in microglia-glioma cell interactions. We now show in human tumor specimens (42 patients), that 39% of tumor-associated microglial (TAM) cells express mTOR phosphorylated at Ser-2448; and similar mTOR activation is observed using a human microglia-glioma interaction paradigm. In addition, we confirm previous studies that microglia express urea and ARG1 (taken as M2 marker) in the presence of glioma cells, and this phenotype is down-regulated in the presence of a mTOR inhibitor. These results suggest that mTOR suppression in GBM patients might induce a reduction of the M2 phenotype expression in up to 40% of all TAMs. Since the M2 profile of microglial activation is believed to be associated with tumor progression, reductions in that phenotype may represent an additional anti-tumor mechanism of action of mTOR inhibitors, along with direct anti-proliferative activities.