Abstract
Of all of the types of primary brain tumors, glioblastoma multiforme (GBM) is both the most common and most aggressive. As a direct result of its aggressiveness and its location within sensitive brain tissue beyond the blood-brain barrier, successful treatment is a significant challenge and mortality is high, even when patients receive optimal treatment. Further, GBM is generally resistant to conventional therapies including surgery, radiation, and chemotherapy. This highlights the necessity for the development of novel therapies to treat GBM, such as the manipulation of host immune cells to specifically target the most tumorigenic and treatment-resistant GBM cells. Evidence continues to accumulate that supports a cancer stem cell (CSC) model. In this model, CSCs are a relatively small subpopulation of cancer cells within a heterogeneous tumor population that are responsible for tumor recurrence and metastatic tumorigenesis. Cells with this phenotype have been observed in many different types of cancers, including GBM tumors, and these CSCs have been shown to exhibit both radioresistance and chemoresistance. For these reasons, GBM stem cells are an attractive target for new therapies that seek to effectively treat GBM. For solid tumors like GBM, natural killer (NK) cells have shown efficacy both in vitro and in vivo in recognizing and killing human tumor cells through specific receptor-ligand interactions that promote apoptosis. Research has found that the induction of cellular stress in tumor cells can sensitize GBM stem cells to this NK cell-mediated killing. Bortezomib, an FDA-approved small-molecule proteasome inhibitor, is currently used for the treatment of hematologic malignancies such as multiple myeloma. Bortezomib is one chemotherapeutic agent that has been shown to sensitize cancer cells, and specifically CSCs, to NK cell-mediated killing via cancer cell upregulation of a combination of death receptors and stress ligands which bind to cognate receptors and ligands on NK cells. Previous research suggests that the cellular stress created by the inability of cancer cells to degrade pro-apoptotic molecules due to proteasome inhibition is one major factor in the sensitization process. The goal of this project was to evaluate whether a combination treatment of bortezomib and ex vivo-activated NK cells would be effective in killing GBM stem cells and whether there would be enhanced killing overall relative to GBM non-CSCs. As part of this goal, we also examined the mechanisms by which NK cells eliminate GBM stem cells. We hypothesized that pre-treating a heterogeneous GBM cell population with bortezomib prior to co-culture with NK cells would enhance NK cell killing of the subpopulation of GBM stem cells, leading to differential killing between the GBM stem cell and GBM non-stem cell populations. Although the main focus was on GBM, we also studied the effects of bortezomib on sarcomas and other cell types to determine whether there was a more generalizable effect. This project had two primary aims: 1. To demonstrate that enhanced CSC-specific killing by ex vivo-activated human NK cells in vitro following bortezomib exposure occurs in GBM, we used flow cytometric analysis to measure cancer cell viability and to identify cell types and expression of surface proteins associated with activation of NK cell cytotoxicity. 2. To identify which mechanisms of cytotoxicity are involved in the dynamic interaction between NK cells and bortezomib-sensitized GBM stem cells. Flow cytometric analysis was again performed to assess cancer cell viability in this altered context. We found that the cellular responses to bortezomib exposure were variable depending on the cell line and tumor type. In U-87 MG glioblastoma cells, the main focus for this project, exposure to bortezomib resulted in increased expression of a CSC marker and increased numbers of putative CSCs. Further, bortezomib exposure resulted in increased cell surface expression of key NK-interacting receptors and ligands in a dose-dependent manner. The Ewing’s sarcoma A673 cell line also shared a similar trend in increased marker expression following bortezomib exposure. Despite an increase in the specific receptors and ligands that activate NK-mediated cytotoxicity, there was also an increase in the expression of an inhibitory surface molecule, which was significantly higher in the CSC subpopulation. This suggests a possible ability of CSCs to evade NK-mediated immunity by producing an overall inhibitory signal. Future work will be necessary to determine whether the inhibition can be overcome to allow successful targeting and destruction of the CSC subpopulation. This project was undertaken at the University of California, Davis Institute for Regenerative Cures in Sacramento, California, under the supervision of the sponsoring mentor, Dr. William Murphy in the Department of Dermatology. The project was completed over the internship period beginning in June 2016 and ending in December 2016.