Human glioblastoma (GBM), the most common and deadly primary human adult brain cancer, remains incurable
with median survivals of 12-20 months. Despite numerous clinical trials, the standard of care temozolomide and
radiation treatment has been unchanged since its introduction in 2005. Arguably, the major contributor to GBM
malignancy is the glioma stem-like cell (GSC). Although a relatively small minority cancer cell population, GSCs
have a disproportionately large role in driving GBM progression by their inherent treatment resistant and diffusely
invasive growth capacity that limit the effectiveness of surgery, radiation and chemotherapy. Conversely, the
larger pool of non-stem glioma cells (NSGCs) are more responsive to standard chemo-radiation and less
invasive. These properties underlie an emerging interest in strategies to reprogram GSCs to less malignant and
more treatment responsive NSGC phenotypes. The role of GSC intrinsic electrical signaling or bioelectricity
(BioE) has fundamental influences on cell states with relevance to therapeutic GSC reprogramming. For
example, BioE signaling is largely regulated through ion channel mediated regulation of voltage membrane
potential (Vmem) whereby depolarization promotes undifferentiated stem-like phenotypes and increased
proliferation while hyperpolarization drives differentiated (i.e., non-stem) and less proliferative states. Here we
test the hypothesis that patterned optogenetic neuro-modulation (PONM) of Vmem can reprogram GSC
phenotypes. Optogenetic techniques provide unprecedented temporal and spatial control of BioE regulation not
possible with genetic, pharmacologic or transcranial electrical stimulation. Using in vitro and in vivo orthotopic
human GSC xenograft models, we will determine how chronicity, intensity, and temporal-spatial modulation of
activating and inhibitory optogenetic channels in GSCs impacts proliferation and stem cell properties. The system
functions through expression of activating channel rhodopsins and inhibitory halorhodopsins, cell cycle sensors,
and novel light delivery platforms that permit facile temporal-spatial regulation of BioE/Vmem and real-time or
post-hoc analysis of GSC phenotypes. Aim 1 will quantify the parameters of PONM that reprogram GSC
phenotypes in vitro while Aim 2 tests the impact of PONM on GSC phenotypes in vivo in GSC xenograft models.
GSC reprogramming will be assessed by quantitation of changes in malignant phenotypes (proliferation, and
invasion) and stem cell properties (self-renewal and quiescence) through a combination of real-time or post-
stimulation assays and gene expression profiling. Stability of reprogrammed phenotypes will be assessed by
delayed in vitro and in vivo analysis after termination of photo-stimulation. The effects of Vmem modulation on
reprogramming GSC xenografts will be measured by histologic, cellular, and molecular profiling analysis of
tumors treated with intratumoral optogenetic stimulation. These studies are expected to provide novel proof of
principle that electrical modulation can reprogram GSC states and support further investigation of targeting
Vmem as a therapeutic adjunct to improve GBM treatment responses and outcomes.