Our goal is to further understand the mechanism by which brain cancer cells, known as glioma cells, killed by prolonged BK ion channel activation can stimulate immune responses to improve immune therapy towards these cancers. Many glioma cells express or over-express these BK ion channels. It is commonly thought that BK channels help make glioma cancers very invasive. Glioma cells killed by prolonged BK channel activation induce cell death via a programmed cell death pathway called paraptosis. Paraptosis is the process that leads to necrosis. This pathway is characterized by a swelling/vacuolization process that ultimately results in osmotic lysis followed by the induction of strong anti-tumor immunity. We hypothesize that paraptotic cells are strongly immunostimulatory, via the release of """"""""danger signals"""""""" that stimulate local dendritic cells (DC) to mature and stimulate effective T cell immunity. We will test this concept using a non- immunogenic F98 rat glioma model with paraptotic T9 glioma cells, as the whole cell vaccine. We hypothesize that a newly described spliced variant of the BK channel found within gliomas, called the glioma BK channel (gBK), has 2 epitopes that can stimulate cytotoxic T lymphocytes (CTLs). This project has 4 aims: 1) To identify the danger signal molecules produced by BK channel activated/killed T9 tumor cells that stimulate dendritic cell maturation;2) To prove that the dendritic cells stimulated by the paraptotic T9 cells can generate T9 glioma-specific T cell immunity;3) To verify that paraptotic T9 cells generate cross protective immunity so that established non- immunogenic F98 glioma are successfully treated;4) To confirm that 2 epitopes derived from the gBK ion channel can induce human HLA-A2+ restricted CTL responses. Previously used glioma models developed by the investigators will be employed for this work. BK channel activated and killed T9 glioma cells die via paraptosis;these killed cells are effective in generating cell mediated immunity against seven day established F98 tumors. A variety of in vitro studies will be done using confocal microscopy, siRNA, flow cytometry, cytolytic assays, recombinant danger signals, and quantitative RT-PCR. Studies in rats will show that effective tumor immunity is generated after the paraptotic glioma cells or danger signals are used as a component in the tumor vaccine. Glioma cells possess both wild-type BK and gBK channels, contrary to a prior report by another group. We have produced a polyclonal antibody that detects gBK. Rat and human glioma cell lines possess gBK as detected with this antibody. Freshly resected GBM tissue express gBK. Human gBK-specific CTLs have killed HLA-A2+ glioma cells making gBK. Hence we are well-positioned to do this project. The way cancer cells die profoundly influences the way the immune system responds to these tumor cells. Apoptotic or autophagic cells lead to immune tolerance, while paraptotic cells produce strong immunity. These proposed studies will delineate a novel mechanism whereby glioma cells stimulate anti-tumor immune responses using whole tumor cells killed by prolonged BK channel activation without any need of genetic manipulation of the cells. Thus, we envision that glioma cell lines, either well-established or fresh neurosphere cultures, could be used for vaccination purposes. The knowledge gained from these studies will allow us to design, evaluate, and choose the best vaccination strategy using BK channel activation for treating human gliomas using paraptotic cells as a potential tumor vaccine. The potential for immediate clinical application is therefore significant.
Glioma is a rare cancer but 85 cases have been diagnosed at the Long Beach V.A.M.C. over the last 24 years. These high-grade gliomas however, remain lethal tumors for which no effective therapy exists. Our animal model provides an experimental system to test various therapeutic options with mM-CSF so that practical clinical protocols can be designed. We have an active collaboration with the clinical scientists at Hoag Cancer Center who can immediately apply the principals we learn here into immediate practical approaches into humans. Since our work was reproduced using the human U251 glioma, we can readily envision this therapy being used in our cancer patients. Preliminary in vitro and in vivo data shows that this approach of using paraptotic cells as a possible vaccine appear promising. Thus, we are confident that this rat glioma model is directly applicable with human gliomas.
