Combination treatment of cytomegalovirus positive glioblastoma with Bortezomib and Temozolomide chemotherapy sensitizes for NK-cell immunotherapy
Glioblastoma (GBM) is a malignant brain tumor, where patients’ survival is ≤14.6 months despite multimodal-therapy. Novel, effective combination-therapies that sensitize to chemotherapy/immunotherapy are pivotal for improving survival. We hypothesize that initiating misfolded-protein response (using the proteasome inhibitor, Bortezomib) in combination with Temozolomide (TMZ)-chemotherapy will increase expression of immunogenic stress-ligands that may sensitize GBM-cells to Natural-Killer (NK) immunotherapy. We aim to investigate efficacy, cell-death mechanisms and determine whether sensitization translates to durable responses in vivo with overall-survival and tolerability as endpoints. Since cytomegalovirus (CMV) is a major factor contributing to immune-evasion in GBM, we will undertake the treatment studies in CMV-positive, patient-derived GBM-xenografts. Thus, prior passaging of the patient-biopsies in vivo to generate sufficient and standardized tumor-xenografts will be required. Our preliminary in vitro findings support the hypothesis and demonstrate that:
1) Bortezomib sensitizes GBM-cells to TMZ-chemotherapy through depletion of MGMT that promotes resistance to TMZ-chemotherapy.
2) Bortezomib sensitizes the GBM-cells to NK-cell immunotherapy by increasing expression of stress-ligands (MICA/ULBPs) that are recognized by a killer-receptor on NK-cells.
3) Pretreatment of glioma-cells with 10-12.5 nM Bortezomib prior to TMZ-treatment or co-culture with NK-cells kill cancer-cells but not normal-human-astrocytes more efficiently compared to TMZ or NK-cell monotherapy.
Intracranial tumor implantations will be using to implant tumor-cells on mice brain. Minimal distress for the animals is expected due to surgery on brain. To overcome this 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 ≥20% body-weight. Animals will be sacrificed by CO2 inhalation and decapitation, previously deemed satisfactory. Upon neurological-sequelae, 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. A new treatment will make an effect on GBM prognosis to not only on an individual but also to the society. The proposed combination-treatment sensitizes GBM-cells to TMZ-treatment and reducing the IC50-dose of TMZ from 250 µM to 125 µM, which will reduce the side-effects of TMZ to the patient and treatment cost. We will utilize n=318 mice (Nod-SCID) for 5 different studies over 2 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 and laboratory-assistant with extensive experience will undertake the surgical procedures, ensuring rapid/safe and reproducible intracranial tumor implantations to minimize animal discomfort. We will utilize a stereotaxic-frame and aseptic-technique for accurate co-ordinates, reproducibility and limited infection. Treatment with drugs and/or immune-cells will commence after confirmed MRI visible tumor to ensure valid results/limit repeated experiments. Longitudinal non-invasive MRI will be used to monitor tumor-growth to ensure statistical robustness.
1) Bortezomib sensitizes GBM-cells to TMZ-chemotherapy through depletion of MGMT that promotes resistance to TMZ-chemotherapy.
2) Bortezomib sensitizes the GBM-cells to NK-cell immunotherapy by increasing expression of stress-ligands (MICA/ULBPs) that are recognized by a killer-receptor on NK-cells.
3) Pretreatment of glioma-cells with 10-12.5 nM Bortezomib prior to TMZ-treatment or co-culture with NK-cells kill cancer-cells but not normal-human-astrocytes more efficiently compared to TMZ or NK-cell monotherapy.
Intracranial tumor implantations will be using to implant tumor-cells on mice brain. Minimal distress for the animals is expected due to surgery on brain. To overcome this 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 ≥20% body-weight. Animals will be sacrificed by CO2 inhalation and decapitation, previously deemed satisfactory. Upon neurological-sequelae, 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. A new treatment will make an effect on GBM prognosis to not only on an individual but also to the society. The proposed combination-treatment sensitizes GBM-cells to TMZ-treatment and reducing the IC50-dose of TMZ from 250 µM to 125 µM, which will reduce the side-effects of TMZ to the patient and treatment cost. We will utilize n=318 mice (Nod-SCID) for 5 different studies over 2 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 and laboratory-assistant with extensive experience will undertake the surgical procedures, ensuring rapid/safe and reproducible intracranial tumor implantations to minimize animal discomfort. We will utilize a stereotaxic-frame and aseptic-technique for accurate co-ordinates, reproducibility and limited infection. Treatment with drugs and/or immune-cells will commence after confirmed MRI visible tumor to ensure valid results/limit repeated experiments. Longitudinal non-invasive MRI will be used to monitor tumor-growth to ensure statistical robustness.