Glioblastoma (GBM) is the leading cause of high fatality cancer arising within the adult brain. Electrotherapeutic approaches offer new promise for GBM treatment by exploiting innate vulnerabilities of cancer cells to low intensity electric fields. This report describes the preclinical outcomes of a novel electrotherapeutic strategy called Intratumoral Modulation Therapy (IMT) that uses an implanted stimulation system to deliver sustained, titratable, low intensity electric fields directly across GBM-affected brain regions. This pilot technology was applied to in vitro and animal models demonstrating significant and marked reduction in tumor cell viability and a cumulative impact of concurrent IMT and chemotherapy in GBM. No off target neurological effects were observed in treated subjects. Computational modeling predicted IMT field optimization as a means to further bolster treatment efficacy. This sentinel study provides new support for defining the potential of IMT strategies as part of a more effective multimodality treatment platform for GBM.

Purpose Application of low intensity electric fields to interfere with tumor growth is being increasingly recognized as a promising new cancer treatment modality. Intratumoral modulation therapy (IMT) is a developing technology that uses multiple electrodes implanted within or adjacent tumor regions to deliver electric fields to treat cancer. In this study, the determination of optimal IMT parameters was cast as a mathematical optimization problem, and electrode configurations, programming, optimization, and maximum treatable tumor size were evaluated in the simplest and easiest to understand spherical tumor model. The establishment of electrode placement and programming rules to maximize electric field tumor coverage designed specifically for IMT is the first step in developing an effective IMT treatment planning system. Methods Finite element method electric field computer simulations for tumor models with 2 to 7 implanted electrodes were performed to quantify the electric field over time with various parameters, including number of electrodes (2 to 7), number of contacts per electrode (1 to 3), location within tumor volume, and input waveform with relative phase shift between 0 and 2π radians. Homogeneous tissue specific conductivity and dielectric values were assigned to the spherical tumor and surrounding tissue volume. In order to achieve the goal of covering the tumor volume with a uniform threshold of 1 V/cm electric field, a custom least square objective function was used to maximize the tumor volume covered by 1 V/cm time averaged field, while maximizing the electric field in voxels receiving less than this threshold. An additional term in the objective function was investigated with a weighted tissue sparing term, to minimize the field to surrounding tissues. The positions of the electrodes were also optimized to maximize target coverage with the fewest number of electrodes. The complexity of this optimization problem including its non‐convexity, the presence of many local minima, and the computational load associated with these stochastic based optimizations led to the use of a custom pattern search algorithm. Optimization parameters were bounded between 0 and 2π radians for phase shift, and anywhere within the tumor volume for location. The robustness of the pattern search method was then evaluated with 50 random initial parameter values. Results The optimization algorithm was successfully implemented, and for 2 to 4 electrodes, equally spaced relative phase shifts and electrodes placed equidistant from each other was optimal. For 5 electrodes, up to 2.5 cm diameter tumors with 2.0 V, and 4.1 cm with 4.0 V could be treated with the optimal configuration of a centrally placed electrode and 4 surrounding electrodes. The use of 7 electrodes allow for 3.4 cm diameter coverage at 2.0 V and 5.5 cm at 4.0 V. The evaluation of the optimization method using 50 random initial parameter values found the method to be robust in finding the optimal solution. Conclusions This study has established a robust optimiza...

Introduction Diffuse intrinsic pontine glioma (DIPG) is a high fatality pediatric brain cancer without effective treatment. The field of electrotherapeutics offers new potential for other forms of glioma but the efficacy of this strategy has not been reported for DIPG. This pilot study evaluated the susceptibility of patient-derived DIPG cells to low intensity electric fields delivered using a developing technology called intratumoral modulation therapy (IMT). Methods DIPG cells from autopsy specimens were treated with a custom-designed, in vitro IMT system. Computer-generated electric field simulation was performed to quantify IMT amplitude and distribution using continuous, low intensity, intermediate frequency stimulation parameters. Treatment groups included sham, IMT, temozolomide (TMZ) chemotherapy and radiation therapy (RT). The impact of single and multi-modality therapy was compared using spectrophotometric and flow cytometry viability analyses. Results DIPG cells exhibited robust, consistent susceptibility to IMT fields that significantly reduced cell viability compared to untreated control levels. The ratio of viable:non-viable DIPG cells transformed from ~ 6:1 in sham-treated to ~ 1.5:1 in IMT-treated conditions. The impact of IMT was similar to that of dual modality TMZ–RT therapy and the addition of IMT to this treatment combination dramatically reduced DIPG cell viability to ~ 20% of control values. Conclusions This proof-of-concept study provides a novel demonstration of marked DIPG cell susceptibility to low intensity electric fields delivered using IMT. The potent impact as a monotherapy and when integrated into multi-modality treatment platforms justifies further investigations into the potential of IMT as a critically needed biomedical innovation for DIPG.

Introduction High‐Grade Gliomas (HGG) are primary malignant brain tumors with dismal treatment outcomes. Our team has been pioneering a novel implantable biotechnology called Intratumoral modulation therapy (IMT) that distributes non‐ablative electric fields across tumor‐affected brain regions to induce tumor cell death. A systematic means to define the mechanism and optimal treatment parameters for various forms of HGG is needed to advance this promising technology towards clinical application. The aim of this study was to develop a custom, high throughput, patient‐derived 3D HGG model to investigate the therapeutic effects of IMT. Methods Primary patient HGG cells (1×104) were implanted into wells of a 96‐well plate containing 200 uL of Matrigel®. The resultant tumor spheroids were characterized over 14 days by observing growth, invasion and viability with MTT assays, confocal microscopy and bioluminescence imaging (BLI). The culture plates were custom adapted to house implantable IMT devices to broadly distribute IMT fields across HGG tumors. Computer‐generated IMT field modeling was performed using COMSOL software to predict field strength and distribution, and to reconstruct IMT fields in the 3D system. Spheroids received 72‐hours of IMT using a spectrum of treatment parameters based upon a continuous, low amplitude sinusoidal waveform. Results Patient‐derived HGG spheroids exhibited multi‐layered, progressive growth and invasion of peritumoral matrix over the 14‐day study period. Computer simulation predicted electric field distribution and intensity across patient HGG spheroids for a spectrum of defined IMT parameters. Using the simulation plans to guide treatment settings, IMT produced a significant reduction (>60%) of metabolic viability in HGG spheroids treated with IMT compared to sham conditions. This data was corroborated by BLI which revealed >65% signal loss associated with IMT. Conclusion IMT is a highly promising innovation designed to combat HGGs, the most devastating of primary brain cancers. Our custom in vitro IMT model permits high throughput testing of treatment parameters in 3D patient HGG spheroids. This innovative preclinical strategy will be instrumental in defining the potential and optimizing treatment response across a spectrum of high fatality HGG cancers. Support or Funding Information This work was supported by the Cancer Research Society and preformed at Schulich School of Medicine and Dentistry and Lawson Health Research Institute.

Objective: The treatment of glioblastoma (GBM) using low intensity electric fields (~1 V/cm) is being investigated using multiple implanted bioelectrodes, which was termed intratumoral modulation therapy (IMT). Previous IMT studies theoretically optimized treatment parameters to maximize coverage with rotating fields, which required experimental investigation. In this study, we employed computer simulations to generate spatiotemporally dynamic electric fields, designed and purpose-built an IMT device for in vitro experiments, and evaluated the human GBM cellular responses to these fields. &#xD;Approach: After measuring the electrical conductivity of the in vitro culturing medium, we designed experiments to evaluate the efficacy of various spatiotemporally dynamic fields: (a) different rotating field magnitudes, (b) rotating vs. non-rotating fields, (c) 200 kHz vs. 10 kHz stimulation, and (d) constructive vs. destructive interference. A custom printed circuit board (PCB) was fabricated to enable four-electrode IMT in a 24-well plate. Patient-derived GBM cells were treated and analyzed for viability using bioluminescence imaging. &#xD;Main results: The optimal PCB design had electrodes placed 6.3 mm from the center. Spatiotemporally dynamic IMT fields at magnitudes of 1, 1.5, and 2 V/cm reduced GBM cell viability to 58%, 37% and 2% of sham controls respectively. Rotating vs. non-rotating, and 200 kHz vs. 10 kHz fields showed no statistical difference. The rotating configuration yielded a significant reduction (p<0.01) in cell viability (47±4%) compared to the voltage matched (99±2%) and power matched (66±3%) destructive interference cases.&#xD;Significance: We found the most important factors in GBM cell susceptibility to IMT are electric field strength and homogeneity. Spatiotemporally dynamic electric fields have been evaluated in this study, where improvements to electric field coverage with lower power consumption and minimal field cancellations has been demonstrated. The impact of this optimized paradigm on cell susceptibility justifies its future use in preclinical and clinical trial investigations.

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Cancer stem-like cells (SLC) resist conventional therapies, necessitating searches for SLC-specific targets. We established that cyclo-oxygenase(COX)-2 expression promotes human breast cancer progression by activation of the prostaglandin(PG)E-2 receptor EP4. Present study revealed that COX-2 induces SLCs by EP4-mediated NOTCH and WNT up-regulation. EP4 antagonist (EP4A) treatment ablated SLCs both in vitro and in vivo. Ectopic COX-2 over-expression in MCF-7 and SKBR-3 human breast cancer cell lines (named MCF-7-COX-2 and SKBR-3-COX-2) resulted in aggressive phenotypes: increased migration/invasion/proliferation, EMT, elevated SLCs, evidenced by spheroid formation for successive generations, increased ALDH activity and co-expression of COX-2/SLC markers. These changes were reversed with COX-2 inhibitor or EP4A, indicating dependence on COX-2/EP4 activities. COX-2 overexpression or EP4 agonist treatment of COX-2 low cells caused up-regulation of NOTCH/WNT pathway genes, blocked with PI3K/AKT inhibitors. Supporting above findings, micro-array analysis showed up-regulation of numerous SLC-regulatory and EMT-associated genes in MCF-7-COX-2 cells. MCF-7-COX-2 cells showed increased orthotopic tumorigenicity and spontaneous multi-organ metastases in NOD/SCID/IL-2Rγ-deficient mice for successive generations with limiting cell inocula. Orthotopic tumors showed significant up-regulation of VEGF-A/C/D, Vimentin and phospho-AKT, down-regulation of E-Cadherin and enrichment of SLC marker positive and spheroid forming cells. MCF-7-COX-2 cells also showed increased lung colonization in NOD/SCID/GUSB-null mice, an effect reversed with EP4 knockdown or EP4A treatment. COX-2, EP4 and ALDH1A expression in situ in human breast cancer tissues were highly correlated with one other, more marked in progressive stage of disease. High COX-2/EP4 expression was linked with poor survival. Thus EP4 represents a novel SLC-ablative target in human breast cancer. (Supported by a grant of the OICR to PKL and a TBCRU fellowship to MM) Citation Format: Mousumi Majumder, Xiping Xin, Ling Liu, Elena Tutunea-Fatan, Mauricio Rodriguez-Torres, Krista Vincent, Andrew Deweyert, Lynne-Marie Postovit, David Hess, Peeyush K. Lala. Breast cancer stem cell induction by COX-2 via EP4/PI3K-AKT/NOTCH-WNT axis: EP4 as therapeutic target. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3315.

Human cancer xenografts are a vital tool for understanding tumor biology, growth kinetics, and therapeutic efficacy using animal models. Historically, immunodeficient mice have been the standard rodent species for cancer xenograft modeling. However, an immunodeficient rat that supports a wide variety of human cancer cell types would provide a larger rodent strain for easier surgical manipulation, serial blood sampling, and provide a single model in which efficacy, pharmacokinetics, and toxicology can be performed. We have created a Sprague Dawley Rag2 -/-, Il2rg -/- rat (SRGTM OncoRat®) that provides a highly supportive environment for growing tumors of human origin. The SRG rat lacks B, T, and NK cells and readily supports the growth of multiple human cancer cell lines. The SRG rat is more immunodeficient than the Nude rat, suggesting it may be permissive to a wider variety of human cancer types. Here we demonstrate the utility of the SRG rat for both subcutaneous and orthotopic xenograft modeling. The SRG rat supports the growth of both lung and liver orthotopic cancers. In addition, the SRG rat supports the growth of orthoptic human glioblastoma multiforme in the brain. We use in vivo imaging to visualize tumor establishment and growth in subcutaneous, orthotopic, and metastatic models. Furthermore, our data show the ability of the SRG rat to support the growth of multiple different human cancer cell types subcutaneously in two different matrices, Matrigel® and VitroGel®. These data confirm that the SRG rat is an excellent host for studying human cancer. Our data demonstrate that the SRG rat has a high utility for studies using in vivo imaging, orthotopic tumor implantation, and standard subcutaneous tumor modeling. As the most immunodeficient rat commercially available, the SRG rat supports the growth of multiple human cancer types in a larger rodent strain relative to commercially available mouse models. Citation Format: Diane Begemann, Aida Javidan, Cynthia Dunn, Nicolas Johnston, R. Grace Walton, Valeriya Steffey, Ian Corbin, Niveen Fulcher, Cleusa De Oliveira, Hu Xu, Mila Uzelac, Andrew Deweyert, John A. Ronald, Susanne Schmid, Matthew O. Hebb, Fallon K. Noto. In vivo subcutaneous and orthotopic cancer xenograft modeling in the SRG immunodeficient rat [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 42.