Identification and functional characterisation of molecular and epigenetic biomarkers of malignant progression in glioblastoma and immune subsets
Glioblastoma (GBM) is the malignant brain tumor, where 90% of patients are cytomegalovirus (CMV) seropositive and overall-survival is less than 15-months, despite aggressive multimodal therapy. Novel, effective therapeutic strategies are sorely needed for improving patient survival. Previously we demonstrated NG2/CSPG4 as an independent biomarker for poor survival where the proteoglycan promoted GBM angiogenesis, resistance to chemo-and-radiotherapy. We recently identified a tumor-specific 13-base pair (bp) deletion in the NG2/CSPG4-gene in patients´ GBM-biopsies that was not present in healthy-controls. We hypothesize that this novel 13-bp deletion mutation in NG2/CSPG4 may affect the biological growth of the tumor, such as proliferation, apoptosis, and angiogenesis in patient-derived-xenograft (PDX) models. One mechanism of temozolomide chemoresistance is upregulation of autophagy, a process whereby cells degrade and recycle misfolded proteins and organelles to sustain themselves during conditions of stress, such as chemotherapy. Combination-treatment of temozolomide with bortezomib, a proteasome inhibitor, that we previously showed decreases MGMT-protein expression and block autophagy, may strongly sensitize the NG2/CSPG4+ chemoresistant GBM-cells to apoptosis. Bortezomib also affects immune cell differentiation, and since CMV is a major factor contributing to immune-evasion in GBM, we will simultaneously investigate whether Natural-killer (NK) cell mediated responses to the CMV+ cancer are affected. NK-cells use killer immunoglobulin receptors (KIRs) to recognize human leucocyte antigen (HLA) ligands and distinguish normal from diseased-cells. NK-cell subsets from CMV+NG2/CSPG4+ patients will be investigated for their ability to efficiently kill GBM in vivo. We aim to investigate whether efficacy and cell-death mechanisms translate to durable-responses and overall-survival in animals in vivo. We will undertake the treatment studies in PDX-models bearing CMV-positive tumors. Thus, prior passaging of the patient-biopsies in vivo to generate sufficiently standardized tumor-xenografts will be required.
Our preliminary in vitro findings support the hypothesis and would like to demonstrate in vivo since treatment-tolerability and overall-survival can only be investigated in animals in vivo.
Intracranial tumor implantations will be used to implant tumor-cells in mouse brain. To achieve minimal distress we will utilize local/post-op analgesia. Gaseous-anesthesia during surgical-procedures will reduce mortality. Humane endpoints include severe neurological sequelae and/or loss of 10% body-weight. Upon neurological-sequelae, animals will be sacrificed by cervical-dislocation, tumors will be harvested and analyzed ex vivo by various cellular/molecular-methods that will be correlated with survival-outcomes.
Replacement, reduction and improvement: In vivo validation is required to determine the translational-relevance which is replacing our in vitro studies. A holistic interrogation of how treatment-effects are modified by the complex in vivo microenvironment of GBM characterized by cellular-heterogeneity, oxygenation/hypoxia, blood-flow and intracranial-pressure will be necessary. Thus the proposed in vivo study aims to confirm our encouraging in vitro findings and establish the modality for future patient-trials. We will utilize n=468 mice (Nod-SCID) for 6-different studies over a 3-years-period. To minimize the number of animals we have used statistical power-analysis for sample size required to distinguish significant effects between treatment-groups. A neurosurgeon with extensive experience will utilize a stereotaxic-frame during surgery and aseptic-technique for accurate co-ordinates, reproducibility and limited infection. Longitudinal non-invasive MRI will be used to monitor tumor-growth to ensure statistical robustness.
Our preliminary in vitro findings support the hypothesis and would like to demonstrate in vivo since treatment-tolerability and overall-survival can only be investigated in animals in vivo.
Intracranial tumor implantations will be used to implant tumor-cells in mouse brain. To achieve minimal distress we will utilize local/post-op analgesia. Gaseous-anesthesia during surgical-procedures will reduce mortality. Humane endpoints include severe neurological sequelae and/or loss of 10% body-weight. Upon neurological-sequelae, animals will be sacrificed by cervical-dislocation, tumors will be harvested and analyzed ex vivo by various cellular/molecular-methods that will be correlated with survival-outcomes.
Replacement, reduction and improvement: In vivo validation is required to determine the translational-relevance which is replacing our in vitro studies. A holistic interrogation of how treatment-effects are modified by the complex in vivo microenvironment of GBM characterized by cellular-heterogeneity, oxygenation/hypoxia, blood-flow and intracranial-pressure will be necessary. Thus the proposed in vivo study aims to confirm our encouraging in vitro findings and establish the modality for future patient-trials. We will utilize n=468 mice (Nod-SCID) for 6-different studies over a 3-years-period. To minimize the number of animals we have used statistical power-analysis for sample size required to distinguish significant effects between treatment-groups. A neurosurgeon with extensive experience will utilize a stereotaxic-frame during surgery and aseptic-technique for accurate co-ordinates, reproducibility and limited infection. Longitudinal non-invasive MRI will be used to monitor tumor-growth to ensure statistical robustness.