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. Novel, effective therapeutic strategies are needed. Previously we demonstrated NG2/CSPG4 as an independent biomarker for poor survival. We recently identified a tumor-specific 13-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, angiogenesis in PDX models. 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. BTZ 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 KIRs to recognize 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 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. As some of the cells are proliferating slower in vitro and we expected to get the tumor in mice slower or may be no tumor with an extended survival, therefore maximum duration of the experiment for the individual animal will be 300 days.
In vivo validation is required to determine the translational-relevance which is replacing our in vitro studies. The proposed in vivo study aims to confirm our encouraging in vitro findings and establish the modality for future patient-trials. We have planned to 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. We could not finish our planned experiments mostly due to time-consuming process of cell generation in the lab. The cell generation process in ongoing and we aimed to start the animal experiments soon. We would like to extend the project for one more year. 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. As some of the cells are proliferating slower in vitro and we expected to get the tumor in mice slower or may be no tumor with an extended survival, therefore maximum duration of the experiment for the individual animal will be 300 days.
In vivo validation is required to determine the translational-relevance which is replacing our in vitro studies. The proposed in vivo study aims to confirm our encouraging in vitro findings and establish the modality for future patient-trials. We have planned to 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. We could not finish our planned experiments mostly due to time-consuming process of cell generation in the lab. The cell generation process in ongoing and we aimed to start the animal experiments soon. We would like to extend the project for one more year. 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.