No abstract

Glioblastoma (GBM) is a deadly malignancy with a poor prognosis. An important factor contributing to GBM recurrence is high resistance of GBM cancer stem cells (GSCs). While temozolomide (TMZ), has been shown to consistently extend survival, GSCs grow resistant to TMZ through upregulation of DNA damage repair mechanisms and avoidance of apoptosis. Since a single-drug approach has failed to significantly alter prognosis in the past 15 years, unique approaches such as multidrug combination therapy together with distinctive targeted drug-delivery approaches against cancer stem cells are needed. In this review, a rationale for multidrug therapy using a targeted nanotechnology approach that preferentially target GSCs is proposed with discussion and examples of drugs, nanomedicine delivery systems, and targeting moieties.

Glioblastoma (GBM) is a malignant brain tumor with a poor long-term prognosis. The current median survival is approximately fifteen to twenty months with the standard of care therapy which includes surgery, radiation, and chemotherapy. An important factor contributing to recurrence of GBM is high resistance of GBM cancer stem cells (CSCs) to several anticancer drugs, for which a systemically delivered single drug approach will be unlikely to produce a viable cure. Therefore, multidrug therapies have the potential to improve the survival time. Currently, only temozolomide (TMZ), which is a DNA alkylator, affects overall survival in GBM patients. CSCs regenerate rapidly and over-express a methyl transferase which overrides the DNA-alkylating mechanism of TMZ, leading to drug resistance. Idasanutlin (RG7388, R05503781) is a potent, selective MDM2 antagonist that additively kills GBM CSCs when combined with TMZ. Nanotechnology is an emerging field that shows great promise in drug delivery and diagnostics. The ability to combine both therapy and imaging allows real time assessment of drug delivery in vivo for the field of theranostics.To develop a multi-drug therapy using multi-functional nanoparticles (NPs) that preferentially target the GBM CSC subpopulation and provide in vivo preclinical imaging capability. Polymer-micellar NPs composed of poly(styrene-b-ethylene oxide) (PS-b-PEO) and poly(lactic-co-glycolic) acid (PLGA) were developed investigating both single and double emulsion fabrication techniques as well as combinations of TMZ and RG7388. The NPs were covalently bound to a 15-base-pair CD133 aptamer in order to target the CD133 antigen expressed on the surface of GBM CSC subpopulation. For theranostic functionality, the NPs were also labelled with a radiotracer, Zirconium-89 (89Zr). The NPs maintained a small size of less than 100 nm, a low negative charge and exhibited the ability to effectively target and kill the CSC subpopulation. In addition, the conjugation of the CD133 aptamer was able to promote killing in CSCs leading to the justification of a targeted nanosystem to potentially improve localized therapy in future in vivo models. This work has provided a potentially therapeutic option for GBM specific for CSC targeting and theranostic imaging.

Hypothesis: Immunotherapies hold great promise for treatment of highly resistant cancers, such as glioblastoma (GBM). We hypothesized that high powered imaging can be effectively combined to quantitatively assess the therapeutic efficacy of human derived natural killer (hNK) cells in an orthoptic xenografted mouse model of GBM. Methods: Tumor take (TT) was established via fluorescence. Mice in the treatment (n=5) and control (n=4) groups were given IV hNK cells and physiological saline, respectively. MRI and PET scans were performed four and six weeks after tumor implantation. Histological slices were taken at time of death. Software analysis of tumor volume, standardized uptake value (SUV), and tumor-to-brain ratio (TBR) was conducted via Qimage and Indica Labs - HALO. Results: Mean growth rates are as follows: T1 volume (mL) – 4.1 in the control group vs 2.3 in treatment. T2 volume (mL) – 6.0 in control vs 2.7 in treatment. PET volume (mL) – 3.1 in control vs 2.1 in treatment, SUV (g/mL) – 5.4 in control vs 3.0 in treatment. Two-tailed t-test analysis showed statistical significance (p < 0.01) in T1 volume data. Conclusion and Potential Impact: Treatment group mice showed a trend in reduction in growth rate of tumor volume and SUV compared to control, with a correlated lower histology TBR, suggesting effective in vivo assessment of hNK therapeutic efficacy in the mouse model of GBM via MR/PET imaging. Future trials should provide a larger population size to increase reliability, precision and power.

Background and Hypothesis: Glioblastoma (GB) is an aggressive primary brain malignancy with a mean survival time of 15-16 months. Initial therapy is limited to surgery, radiation and temozolamide (TMZ) chemotherapy with up to seventy percent of GBs recurring in the first year and only five percent of patients still alive after five years. A reason these tumors are difficult to treat is the effect that the GB tumor microenvironment has on recurrence. One major cell type in this microenvironment is GB cancer stem cells and these cells play a major role in recurrence of the GB tumor. Our approach is that if we reduced tumor volume with the standard of care and treat with NK-cell based treatment and combination drug therapy we can improve the remission and prognosis of GB tumors. Experimental Design or Project Methods: To explore the killing potential of NK cells as well as combination drug treatment we set up a caspase assay that would indicate DNA damage and cell health. We set up in a 96-well plate with GB43 or Cancer stem cells with increasing consideration of either drug over 72 hours or NK cells over 48 hours. After we create, dose curves to find LD50 of both Cell based and drug based treatment. Results: We are still assessing the cell-based therapy and calculating LD50 for both treatments. However, we have found that in the drug based combination therapy we were able to show that paclitaxel and RG3788 both alone and in combination with the standard of care,TMZ, showed significantly reduced GB43 cell viability as concentration increased at the 72 hour time point. However, cancer stem cell remain at around 80% viability across all drug combinations and concentrations. Conclusion and Potential Impact: In conclusion at this point these data indicate that GB43 with the standard of care TMZ in combination with paclitaxel and RG3788 have the ability to kill cells in vitro. Moving forward we plan to package paclitaxel and RG3788 in nanoparticles for more efficient targeting of cells to target tumors. In addition, we plan to create lethal dose curves for NK cells and control neuronal cell tissue to better assess drug toxicity and efficacy.