P13.12 Effect of tumor treating fields on tumor microtubes in glioma

BACKGROUND Tumor microtubes (TMs) are ultralong membrane protrusions of tumor cells in astrocytic gliomas, including glioblastomas. TMs are used as routes for brain invasion and for cells to interconnect over long distances resulting in a functional network that allows multicellular communication. This network mediates resistance against the cytotoxicity of radiation and chemotherapy. One explanation for TM network protection is a better homeostasis of calcium ions that would otherwise increase to toxic intracellular levels in response to these therapies. Our working hypothesis is that interfering with the integrity of the glioblastoma cell network is key to a potential breakthrough in glioma therapy. Many cellular structures are polarized and composed of charged elements and are thus potential subjects to electrical forces; this might also influence the complex intercellular calcium waves (ICWs) that are characteristic for glioma networks. We were therefore interested in the effect of TTF on glioma network maintenance. MATERIAL AND METHODS To examine the effect of TTF on glioma TMs we have established a 2D in vitro glioma model using glioblastoma stem cells (GBSCs) grown in high-glucose medium and a 3D model using glioma tumor organoids. Both models reliably reproduce functionality and complexity of morphological features we observe in our mouse model. We analyzed the disruption of tumor network complexity and disruption of functionality by measuring intercellular calcium waves. Tumor cell death and proliferation was investigated in the 2D in vitro glioma model using the inovitroTM-System. RESULTS A peculiar “cricked-TM” phenotype that rarely (0.2% ±0.14) occurred under standard or control conditions was observed in TTF-treated cells (16.22% ±5.12). Cell number was reduced by 75% in two lines of GBSCs after 5 days of TTF exposure; predominantly TM-rich GBSCs (> 4 TMs) were affected. This reduction in tumor cell number corresponded with an increase in cell death (0.3% ±0.09 in untreated cells; 1.4% ±0.45 at day 5 of TTF exposure). The frequency of intercellular calcium transients, a measurement for calcium wave frequency in the glioma networks, was instantly reduced after TTF exposure to 58% ±20.42 of control levels in the primary GBSC 2D culture, and to 57.78% ±12.34 in tumor organoids derived from 3 glioblastoma patients. CONCLUSION This data suggests a potential effect of TTF application on tumor cell networks, at least in vitro. Interestingly, particularly those glioblastoma cells that have so far been proven to be resistant to radio- and chemotherapy appeared to be affected. We will confirm the observed effects of TTFs on tumor cell calcium signaling in our in vivo chronic cranial window mouse model. We anticipate that the results of our project will provide important insights into the underlying mechanism of TTF therapy.

Elevated cellular PpIX potentiates sonodynamic therapy in a mouse glioma stem cell-bearing glioma model by downregulating the Akt/NF-κB/MDR1 pathway

Glioblastoma (GBM) has high mortality rates because of extreme therapeutic resistance. During surgical resection for GBM, 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is conventionally applied to distinguish GBM. However, surgical intervention is insufficient for high invasive GBM. Sonodynamic therapy (SDT) combined with low-intensity ultrasonication (US) and PpIX, as a sonosensitizer, is an emerging and promising approach, although its efficacy is limited. Based on our previous study that down-regulation of multidrug resistant protein (MDR1) in GBM augmented the anti-tumor effects of chemotherapy, we hypothesized that elevation of cellular PpIX levels by down-regulation of MDR1 enhances anti-tumor effects by SDT. In high invasive progeny cells from mouse glioma stem cells (GSCs) and a GSC-bearing mouse glioma model, we assessed the anti-tumor effects of SDT with a COX-2 inhibitor, celecoxib. Down-regulation of MDR1 by celecoxib increased cellular PpIX levels, as well as valspodar, an MDR1 inhibitor, and augmented anti-tumor effects of SDT. MDR1 down-regulation via the Akt/NF-κB pathway by celecoxib was confirmed, using an NF-κB inhibitor, CAPÉ. Thus, elevation of cellar PpIX by down-regulation of MDR1 via the Akt/NF-κB pathway may be crucial to potentiate the efficacy of SDT in a site-directed manner and provide a promising new therapeutic strategy for GBM.

HGG-40. FOCUSED ULTRASOUND ENHANCES ETOPOSIDE DELIVERY IN A MURINE PONTINE GLIOMA MODEL

Background Diffuse intrinsic pontine glioma (DIPG) is a devastating pediatric brain cancer with limited treatment options and poor survival. The delivery of systemic therapies in this disease is severely limited by the blood-brain barrier (BBB). Focused ultrasound combined with intravenous microbubbles (FUS+MB) can effectively open the BBB permitting the entry of drugs across the cerebrovasculature. Etoposide is a chemotherapy frequently used in pediatric oncology with well-established anti-tumor effects but limited efficacy when administered systemically in DIPG. Given that FUS+MB in DIPG is not well studied, our goal was to determine the feasibility of ultrasound-mediated BBB opening and etoposide delivery in a preclinical murine pontine glioma model. Methods A syngeneic, orthotopic model was established by stereotactic injection of PDGF-B+PTEN-/-p53-/- murine glioma cells into the pons of B6 albino mice. Mice were randomly divided into control (n=6) or FUS+MB groups (n=6). A single-element, spherical-segment FUS transducer (center frequency=1.5MHz) driven by a function generator through a power amplifier was used with concurrent microbubble injection to sonicate the tumor and its margins on post-injection day 14. Immediately after treatment, 5 mg/kg of intraperitoneal etoposide was administered to all 12 mice. All animals underwent cardiac puncture and blood sampling, followed by transcardiac perfusion and brain harvesting. Liquid chromatography-mass spectometry was performed on both serum and tumor tissue to measure etoposide levels. Results Contrast-enhanced MRI demonstrated successful BBB opening in all FUS+MB mice. Compared to control (mean=20.98ng/g), etoposide concentration in the sonicated tumor tissue (mean=164.77ng/g) was nearly eight times greater. Lastly, the mean brain tumor–to-serum ratio was more than fivefold higher in the treated mice (1.50%) compared with the control mice (0.28%) (P<0.005). Conclusions FUS+MB is hereby shown successful in BBB opening and enhanced delivery of etoposide in a preclinical pontine glioma model.