The Feasibility of Enhancing Susceptibility of Glioblastoma Cells to IRE

Using a Calcium Adjuvant

ELISA M. WASSON ,1,4 JILL W. IVEY,2,3 SCOTT S. VERBRIDGE,2,3 and RAFAEL V. DAVALOS1,2,3,4

1Department of Mechanical Engineering, Virginia Tech, Goodwin Hall, 635 Prices Fork Road – MC 0238, Blacksburg,VA 24061, USA; 2Department of Biomedical Engineering and Mechanics, Virginia Tech, 325 Stanger Street, Blacksburg,VA 24061, USA; 3Virginia Tech – Wake Forest University, School of Biomedical Engineering & Sciences, Virginia Tech,325 Stanger St., Blacksburg, VA 24061, USA; and 4Bioelectromechanical Systems Laboratory, Department of Biomedical

Engineering and Mechanics, Virginia Tech – Wake Forest University, School of Biomedical Engineering & Sciences, 325 StangerSt., Blacksburg, VA 24061, USA

(Received 15 April 2017; accepted 16 August 2017)

Associate Editor Dan Elson oversaw the review of this article.

Abstract—Irreversible electroporation (IRE) is a cellularablation method used to treat a variety of cancers. IRE worksby exposing tissues to pulsed electric fields which cause cellmembrane disruption. Cells exposed to lower energies becometemporarily permeable while greater energy exposure results incell death. For IRE to be used safely in the brain, methods areneeded to extend the area of ablation without increasingapplied voltage, and thus, thermal damage. We presentevidence that IRE used with adjuvant calcium (5 mM CaCl2)results in a nearly twofold increase in ablation area in vitrocompared to IRE alone. Adjuvant 5 mMCaCl2 induces deathin cells reversibly electroporated by IRE, thereby lowering theelectric field thresholds required for cell death to nearly halfthat of IRE alone. The calcium-induced death response ofreversibly electroporated cells is confirmed by elec-trochemotherapy pulses which also induced cell death withcalcium but not without. These findings, combined with ournumerical modeling, suggest the ability to ablate up to 3.29larger volumes of tissue in vivo when combining IRE andcalcium. The ability to ablate a larger volume with loweredenergies would improve the efficacy and safety of IRE therapy.

Keywords—Electrochemotherapy, Ablation volume, Finite

element modeling, Irreversible electroporation, Brain cancer,

Combined therapy.

INTRODUCTION

Grade IV astrocytoma, also known as glioblastoma(GBM), is among the most aggressive cancers. Standard

therapies such as surgical resection, radiation, andchemotherapy aim to eliminate the primary tumor in thehopes of alleviating neurological symptoms. Even withstate-of-the-art treatment, the increase in median survivaltime is merely 14 months.39,40

As an alternative to standard treatments, energy-based therapies that utilize electric fields with highmagnitudes (400–3000 V/cm), short durations (100 ls)and low frequencies (~1 Hz) are being used to induce celldeath and enhance drugdelivery. These therapies rely ona phenomenon known as electroporation, which occurswhen an applied electric field causes the transmembranepotential of the cell membrane to rise above a thresholdlimit (~200–1000 mV). The rise in transmembranepotential results in the formation of nanoscale defects,allowing otherwise impermeant ions and molecules toenter. At lower voltages, this permeabilization processmay allow for membrane recovery with removal of theapplied field (reversible electroporation), while celldeath through loss of homeostasis occurs at a highervoltage threshold (irreversible electroporation).27

Irreversible electroporation (IRE) has been shown totreat spontaneous gliomas in canine patients16,17 whilesparing blood vessels and extracellular matrix.35 An IREtreatment consists of delivering electrical pulses through twoneedleelectrodes thatare inserted into thebulktumor.2Sinceelectrical pulses are delivered at short duration and low fre-quencies, this causes minimal heating of the tissue, therebyensuring ablation while mitigating thermal damage.9 IREtreatment results in a sharp delineation between treated anduntreated tissue with submillimeter resolution both in vivo11

and in vitro,3 making it possible to develop finite elementsimulations for predicting lesion volumes before treatment.

Address correspondence to Elisa M. Wasson, Bioelectrome-

chanical Systems Laboratory, Department of Biomedical Engineer-

ing and Mechanics, Virginia Tech – Wake Forest University, School

of Biomedical Engineering & Sciences, 325 Stanger St., Blacksburg,

VA 24061, USA. Electronic mails: ewasson1@vt.edu, jivey@vt.edu,

sverb@vt.edu, davalos@vt.edu

Annals of Biomedical Engineering (� 2017)

DOI: 10.1007/s10439-017-1905-6

� 2017 Biomedical Engineering Society

Reversible electroporation can be achieved usinglower voltage pulse parameters that cause permeabi-lization of the membrane, allowing molecules to enterthe cell, while permitting subsequent recovery. Thistechnique has been used in gene transfection,30 blood–brain barrier disruption7 and drug delivery.21 Elec-trochemotherapy (ECT) utilizes reversible electropo-ration to enhance transport of cell impermeantchemotherapy drugs,21 which has shown to be effectivein treating brain tumors.1,29,36

Because ECT uses lower applied electric field mag-nitudes and fewer pulses than IRE,18 and the extent ofelectroporation largely depends on pulse duration andnumber,5 the zone of treatment is limited. IRE is moreversatile, enabling control of the ablation zone in tis-sue. IRE is effective as a stand-alone therapy forablating the primary tumor without the need forchemotherapy drugs, yet still induces reversible elec-troporation further away from the electrodes, makingcells susceptible to adjuvant therapies. Neal et al.,confirmed a 2–39 larger zone of cell death in vitrousing IRE treatment in combination with chemother-apeutic drugs compared to IRE treatment alone.31

Our hypothesis is that IRE efficacy may be im-proved when combined with adjuvant calcium, for atreatment that is safer than combined treatment withchemotherapeutics. This hypothesis is motivated byresults which demonstrate that ECT pulses used inconjunction with calcium cause more cell death and agreater decrease in cellular ATP than electroporationalone.14 Frandsen et al., hypothesized that this may bedue to ATP depletion resulting from calcium ATPasepumps in the plasma membrane going into overdriveto pump calcium out of the cell, although furtherinvestigation is needed to confirm this mechanism andrule out others.

The motivation for adjuvant calcium combined withIRE is based on the knowledge that electric fieldmagnitude during an IRE treatment decreases as youtravel away from the electrodes. A high electric fieldmagnitude will develop close to the electrodes (irre-versible electroporation) and a low electric field mag-nitude far from the electrodes (reversibleelectroporation). Cells in the irreversibly electropo-rated zone will die through loss of homeostasisresulting from electroporation, while the influx ofcalcium will exacerbate cell death in the reversiblyelectroporated zone. Calcium IRE may accentuate thetreatment margin without applying additional energy.Furthermore, it may provide an advantage over mi-crowave and radiofrequency ablation since the mech-anism is non-thermal and spares vital structures.Though efforts have been made to extend the marginof energy based treatments, to our knowledge, we are

the first to investigate IRE pulses in combination withcalcium.

To test our hypothesis, we cultured glioblastomacells in 3D collagen scaffolds and tested ECT and IREpulses in combination with two concentrations ofCaCl2 solution. The electric field thresholds calculatedfrom in vitro results were then used to inform anumerical model that simulates an in vivo treatmentwith the purpose of predicting treatment volumes.These results suggest that using IRE with a calciumadjuvant enhances lesion size without increasing ther-mal damage.

MATERIALS AND METHODS

Cell Culture

U251 malignant glioma (MG) cells (Sigma Aldrich)were cultured in Dulbecco’s Modified Eagle Medium(Life Technologies) containing 10% fetal bovine serum(Atlanta Biologicals), 1% penicillin/streptomycin (LifeTechnologies) and 0.1% non-essential amino acids(Life Technologies). Cells were routinely passaged at70–90% confluence and kept in a humidified incubatorat 37 �C and 5% CO2. Prior to fabricating the 3Dcollagen scaffolds, cells were removed from their flaskusing trypsin (Life Technologies) and centrifuged at1209g for 5 min. Cells were re-suspended in freshmedium and added to the collagen solution for a finalconcentration of 1 9 106 cells/mL.

Collagen Scaffold Fabrication

3D cell cultures are now recognized as moreappropriate tumor models than 2D monolayer cul-tures.13 This technique has been used previously byArena et al.3 and Ivey et al.24 to study the effects ofIRE on different tumor cell lines using similar matrixcomposition and stiffness. Concentrated collagen stocksolutions (10 mg/mL) were created using rat tail col-lagen type I as described previously.3 While the brainconsists of relatively low amounts of fibrous proteins,collagen provides a convenient scaffold material thatproduces relevant 3D geometry, integrin engagementwith surrounding extracellular matrix, and appropriatecell–cell interactions. Collagen stock solution wasmixed with 109 DMEM (10% of total solution vol-ume) and 1 N NaOH (2% of total collagen volume)until homogenous and adjusted to obtain a pH of 7.0–7.4. Cells in media were mixed into the collagen solu-tions to produce a final collagen concentration of5 mg/mL. Collagen was injected into Polydimethyl-siloxane (PDMS) wells of controlled geometry (10 mmdiameter, 1 mm height) to ensure uniformity of the

WASSON et al.

electric field distribution across experiments. Injectedcollagen was molded flat in the PDMS wells and placedin the incubator to polymerize at 37 �C and 5% CO2

for 20 min. Fresh media was added to the wells andthey were cultured in the incubator for 24 h beforetreatment. The electrical conductivities of the gel-cellmixtures were measured with a conductivity meter toensure similar electrical properties. Collagen hydrogelswithout cells had a conductivity of 1.08 ± 0.06 S/m(n = 4). Collagen hydrogels with cells seeded in thebulk had a conductivity of 1.17 ± 0.08 S/m (n = 4).

Electroporation Protocol

Concentrations of 1 mM and 5 mM CaCl2 wereused in our study to determine the effect of a range ofCaCl2 concentrations on lesion size in vitro. Theseconcentrations have shown effect on cell viability.14

Media was aspirated from each well and a concentra-tion of 1 mM or 5 mM CaCl2 in HEPES buffer wasadded for 30 min at room temperature to ensurecomplete diffusion into the collagen scaffold. Calciumsolutions were then aspirated from each well andscaffolds washed with new CaCl2 solution to ensure allcell culture media had been replaced. Fresh calciumsolutions were added immediately prior to pulsing. Acontrol solution of NaCl in HEPES buffer was used, asit has similar conductivities and osmotic concentra-tions to the CaCl2 solutions (Table 1). All solutionswere within the isotonic range of 260–320 mOsm/Land had pH values between 7.0 and 7.2 to betteremulate a slightly acidic tumor microenviron-ment.8,19,23 Sham treatments were carried out by add-ing the NaCl and CaCl2 solutions to each well andinserting the electrodes without pulsing.

Hollow stainless steel, blunt tip needles (HowardElectronic Instruments) with diameters 0.914 mm(OD) and 0.635 mm (ID) were used as electrodes. Acustom-made part housed the electrodes to ensureuniform spacing (4 mm center-to-center) and place-ment in each collagen scaffold (Fig. 1). IRE pulseswere delivered using an ECM 830 pulse generator(Harvard apparatus) and consisted of eighty 450 Vpulses, frequency of 1 Hz and pulse duration of 100 ls.ECT pulses consisted of the same parameters except

eight pulses were delivered. After treatment, CaCl2 andNaCl solutions were removed from each well, replacedwith cell culture media and the well plate was returnedto the incubator.

Determining Area and Cell Death Electric FieldThresholds in Collagen Scaffolds

Scaffolds were kept in the incubator for 24 h aftertreatment. It has been reported that this is sufficient timeto allow transient pores formed in the cell membrane torecover6 and evaluate the effects of calcium electropora-tion.14,22 Scaffolds were incubated with 2 lM CalceinAM (Invitrogen) and 15 lM propidium iodide (LifeTechnologies) in PBS for 30 min at room temperature.Calcein AM labels living cells green while propidium io-dide labels cells lacking membrane integrity red. Imageswere taken of each well using an inverted DMI 6000Bmicroscope (LeicaMicrosystems) with a95 objective. Acustom algorithm developed in MATLAB was used tomeasure lesion area (see Supplementary Material fordetails). Details describing the numerical model simu-lating collagen scaffold experiments using COMSOLMultiphysics 5.3 (Stockholm, Sweden) and calculation ofelectric field thresholds have been previously reported3,24

and are described in Supplementary Material.

Numerical Model to Simulate Lesion Volumes in theBrain

A simplified finite element model of the brain wascreated using COMSOL Multiphysics 5.3 to simulatethe increased lesion size that would occur in vivo usingIRE and calcium. The brain was modeled as a 3Ddomain with dimensions sufficiently large to mitigateany boundary effects that may influence lesion size andshape (12 cm 9 12cm 9 12cm). This assumption pro-vided us with an ideal case to separate the effectivenessof treatment from any influences geometry andboundary conditions may have on lesion size. Twostainless steel, cylinder electrodes (1 mm diameter),spaced 2.0 cm apart (center-to-center) with a length of1.5 cm were inserted into the center of the 3D domain.The governing equation that determines potential dis-tribution in a material is defined below:

TABLE 1. Properties of solutions used in experiments.

Concentration Solution Conductivity (S/m) Osmolarity (mOsm/L)

1 mM CaCl2 0.053 ± 0.017 276.5 ± 2.12

NaCl 0.106 ± 0.026 282.5 ± 3.53

5 mM CaCl2 0.078 ± 0.005 288.0 ± 9.90

NaCl 0.122 ± 0.009 290.5 ± 4.95

The Feasibility of Enhancing Susceptibility of Glioblastoma Cells

@u@t

¼ r � rruð Þ ð1Þ

Here, u is electric potential and r is electrical con-ductivity of the brain. We were unable to assumeconstant conductivity since it has been shown thatconductivity of tissue increases more dramatically afterelectroporation than cells in suspension due to thelarge ratio of cell volume to extracellular fluid vol-ume.25 Ions leak out of the cells when they are elec-troporated38 thereby increasing conductivity. Jouleheating effects also increase conductivity and act as avolumetric heat source. To model this change in con-ductivity, we employed a smoothed Heaviside functionthat is dependent on electric field.16 We can indirectlyrelate the change in conductivity to the applied electricfield since the extent to which the tissue is electropo-rated depends on this magnitude. Conductivity alsodepends on the change in temperature that occurs inthe tissue, so an additional linear heating term is used.Here, a is the coefficient that describes how conduc-tivity changes with temperature (3.2% �C2110 and T0 isthe initial temperature of the tissue (37 �C). To ensurethe solution will converge, we smoothed the functionusing a continuous second derivative. The functionused to define conductivity is shown below as it waswritten in COMSOL.

r E;TðtÞð Þ ¼ r0 1þ 2 � flc2hs Enorm � Edelta;Erange

� ��

þ aðT tð Þ � T0Þ�ð2Þ

Here Enorm is the magnitude of the electric field,Erange is the range over which the function transitions

(±120 V/cm), Edelta is the electric field value at whichthis transition occurs (580 V/cm) and r0 is a baselineconductivity value for grey matter (0.285 S/m).10,16 In

using this function, we assumed that conductivityincreases by a factor of three since this was reportedfor other organs during electroporation.38 We mustalso account for metabolic heat generation, conduc-tion, and convection due to blood perfusion. We in-cluded the Penne’s Bioheat equation with a modifiedJoule heating term to account for resistive losses.26

qcp@T

@t¼ r � krTð Þ � xbcbqb T� Tað Þ þ q

000 þ r ruj j2� ds

ð3Þ

Here q and cp are the density and specific heat ofbrain tissue respectively, k is thermal conductivity ofbrain tissue, xb is blood perfusion rate, cb and qb arethe specific heat and density of blood, Ta is the arterial

temperature (37 �C), q000is the metabolic heat genera-

tion, r is electrical conductivity of the brain, and u iselectrical potential. We approximated the total resis-tive heating that occurs with a treatment using a dutycycle approach, as opposed to iterating over eachpulse, to reduce computation time while stillaccounting for the heat generated during the total ‘‘ontime’’ of the treatment. Here d is the pulse duration(50 ls) and s is the pulse period (1 s). For treatments inthe brain, it is preferred to use 50 ls pulses as opposedto 100 ls to mitigate thermal damage.16 The surface ofone electrode was treated as energized, one groundedand remaining boundaries as insulated. 3000 V wassimulated giving an applied electric field magnitude of1500 V/cm which is typical of IRE treatments deliv-ered in the brain.16,17 1000 and 2000 V were also testedto establish a relationship between applied voltage andlesion volume. All boundaries were treated as ther-mally ð@T=@n ¼ 0Þ insulative to account for the case ofmaximum heating in the tissue. Material propertieswere defined as stainless steel for the electrodes andgrey matter for brain tissue (Table 2).2,4,10,41,42 Stan-

1000

600

400300200100

500

900800700

(V/cm)(a) (b) (c)

potential

ground

FIGURE 1. Experimental setup. (a) Custom made electrodes inserted into the collagen scaffold platform prior to pulsing withelectrode spacing of 4.0 mm (scale bar 4 mm). (b) Geometry of the finite element model used to simulate treatment of the hydrogelplatform (scale bar 2 mm). The extra fine mesh consisted of 103,297 tetrahedral elements. (c) Electric field distribution in thescaffold after an IRE treatment (450 V, 1 Hz, 100 ls pulse width, 80 pulses) showing the region of collagen affected by differentelectric field thresholds (scale bar 2 mm).

WASSON et al.

dard IRE and ECT treatments were simulated in thenumerical model of the brain and the electric fieldcontour corresponding to its in vitro treatmentthreshold was located. The volumes within theseboundaries were numerically integrated. It wasassumed that homogenous concentrations of CaCl2and NaCl solutions would be achieved in vivo. Themesh was refined until there was less than a 2.0%change in calculated volumes.

Statistical Analysis

All IRE, ECT and sham conditions were tested tenor eleven times (n = 10, n = 11) as determined by apower analysis (Fig. 2). The discrepancy between theserepetitions resulted from collagen detachment duringtreatment. Statistical analyses were performed with aconfidence level of a = 0.05 (JMP Pro 13). Two-wayANOVA was used to test for differences in cell deatharea and cell death threshold. Tukey post hoc com-parisons were used to examine differences amongtreatment groups. Results are shown as arithmeticmean ± standard deviation.

RESULTS

Experimental Setup and Numerical Model SimulatingIRE pulses in Collagen Scaffolds

Figure 1 shows our experimental platform. Theelectrode housing enabled precise positioning of elec-trodes into the collagen scaffold, eliminating variabil-ity that may occur in the electric field distribution dueto varying exposure lengths or boundary effects if theelectrodes were not centered.

Our numerical model was used to predict the fielddistribution in the collagen scaffolds during a typicaltreatment. The experimental platform enabled us tovisualize a range of electric field magnitudes as op-posed to a single value that is applied when testing cells

in suspension using plate electrodes (Fig. 1c). Thisrepresents what occurs in vivo during a typical IRE orECT treatment since cells in the tumor will experiencea gradient of field magnitudes depending on their dis-tance from the electrodes. For ECT treatments, re-versible electroporation occurs at an electric fieldmagnitude around 300 V/cm whereas IRE occursabove 500–600 V/cm.30 In our numerical model of thecollagen scaffolds, these zones can be distinctly visu-alized. By bounding regions of different electric fieldmagnitudes using a contour plot, the model allowed usto determine the field threshold that causes an equiv-alent area of cell death for each of our treatments. Thishighlights our ability to precisely predict ablation sizes.

Experimental Results

Our experimental results confirmed our hypothesisthat calcium IRE results in larger lesions than IREalone. Sham treatments resulted in no cell deaththerefore confirming that the CaCl2 and NaCl solu-tions themselves do not affect cell viability (Fig. 2).Similarly, treating with ECT pulses and NaCl solutionsdid not result in cell death. Due to the small number ofpulses used in an ECT treatment, the cells becomepermeabilized, but subsequently recover.

On the other hand, when we applied IRE pulses inthe presence of NaCl solutions, we see a small region ofdead cells formed in the collagen scaffold. This areaexperiences a higher electric field magnitude for alonger duration of time, therefore electroporating thecells to a greater extent and rendering them incapableof recovering from a loss of homeostasis.

Since NaCl and CaCl2 solutions have comparableconductivities, it can be concluded that the larger lesionsobserved for CaCl2 treatments are due to the action ofcalcium and not additional heating effects that mayoccur due to the conductivities of our buffer solutions.

It should be noted that in some of our experimentalimages, the propidium iodide staining is difficult to see.

TABLE 2. Material properties used in the in vivo numerical model of the brain.

Material Property Value References

Brain tissue Thermal conductivity (k ) 0.565 W m21 K21 9

Specific heat capacity (cp) 3680 J kg21 C21 9

Density (q) 1039 kg m23 9

Metabolic heat generation (q000) 10,437 W m23 30

Temperature coefficient 0.032 �C21 9

Electrical conductivity (r0) 0.285 S m21 9

Blood Specific heat capacity (cp) 3850 J kg21 C21 30

Density (q) 1060 kg m23 29

Perfusion rate (wbÞ 7.15E23 29

Stainless steel electrodes Electrical conductivity (rÞ 2.22E6 S m21 2

Thermal conductivity (k ) 14.9 W m21 K21 4

Specific heat capacity (cp) 477 J kg21 C21 4

The Feasibility of Enhancing Susceptibility of Glioblastoma Cells

This effect has also been seen in previous experimentsusing the same platform24 and although the exactmechanism is not known, it seems as if the cells may bebroken up and are unable to take up and maintain thedye 24 h after treatment. Since Calcein AM only flu-oresces when in the presence of intracellular esterases,we can conclude that cells in the dark region are deadand those stained green with Calcein AM are alive.

Combining all area measurements from the imageprocessing algorithm shows that 1 mMand 5 mMCaCl2treatments lead to an increase in lesion size of nearlydouble that of their respective NaCl controls when usingIRE pulses (Fig. 3). The 1 mM CaCl2 treatment com-bined with IRE, resulted in an average lesion area of25.1 ± 3.9 mm2 whereas its NaCl control resulted in anaverage lesion area of 14.4 ± 2.9 mm2. Combinatorialtreatment using 5 mMCaCl2 solution with IRE resultedin an average lesion area of 32.5 ± 2.0 mm2 whereas5 mM NaCl combined with IRE resulted in an averagelesion area of 13.2 ± 3.6 mm2. Comparing 1 and 5 mMCaCl2 concentrations in combination with IRE resultedin a significant difference in lesion area. There was nosignificant difference between 1 and 5 mM NaCl treat-ments (p = 0.9553).

For ECT pulses, it is evident that calcium treat-ments also had a significantly larger lesion area thantheir NaCl controls for both 1 and 5 mM CaCl2. Le-

sion areas were not significantly different for 5 and1 mM CaCl2 treatments (p = 0.1926). Data for bothNaCl concentrations are not shown in Fig. 3 for ECTtreatments since these pulses did not result in a lesionand were treated as having zero area.

When comparing ECT pulses to IRE pulses, for the1 mM CaCl2 solution, there was no significant differ-ence between lesion areas (p = 0.9451) whereas for the5 mM CaCl2 solution, there was a significant differ-ence. As mentioned previously, an IRE treatment of 80pulses leads to an average lesion area of32.5 ± 2.0 mm2, whereas an ECT treatment of eightpulses lead to an average lesion area of26.7 ± 2.1 mm2 for the 5 mM CaCl2 solution.

Calculating Electric Field Threshold Required for CellDeath

From our numerical model of the collagen scaffold,we determined a relationship between lesion area andelectric field magnitude by performing numerical inte-gration on the surface of the scaffold for a range ofelectric field magnitudes (100–1500 V/cm). We con-structed a curve and fit this data using least squaresfitting in MATLAB. This resulted in a sixth orderpolynomial equation shown in Fig. 4a. Despite thisequation not having relevance to the physics in our

Sham

ECT

IRE

1 mM CaCl2 1 mM NaCl 5 mM CaCl2 5 mM NaCl

FIGURE 2. CaCl2 treatments produce larger cell death lesions than NaCl controls for both IRE and ECT pulses (scale bar 1 mm).Collagen scaffold experiments consisted of 4.0 mm electrode spacing, 100 ls pulse width, 1 Hz frequency and 450 V. IRE treat-ments were delivered with a total of 80 pulses (n 5 11) while ECT treatments were delivered with 8 pulses (n 5 11, n 5 10). 24 hafter treatment, hydrogels were stained with Calcein AM (stains live cells green) and propidium iodide (stains dead cells red).

WASSON et al.

model, we were able to use it to accurately back out theelectric field thresholds for each treatment conditionwithout needing to manually determine them usingCOMSOL. Least squares fitting resulted in a maxi-mum relative error of 4.7%.

Increasing the applied voltage from 450 to 800 Vdemonstrates that it is possible to increase the lesion

area by applying a higher voltage during treatment,however, the applied voltage in our experiment waslimited by the size of the scaffold. If we were to apply800 V in combination with 5 mM CaCl2, the lesion sizeand shape may be influenced by the electrically insu-lated boundaries of the collagen scaffold and anaccurate area may be difficult to measure. An applied

*** ****** ***

FIGURE 3. Lesion sizes measured in collagen scaffold experiments demonstrate that CaCl2 treatments lead to significantly largerlesion sizes than their NaCl controls. Collagen scaffold experiments consisted of 4.0 mm electrode spacing, 100 ls pulse width,1 Hz frequency and 450 V. For IRE treatments, 80 pulses were delivered while for ECT treatments, 8 pulses were delivered.Scaffolds had a total area of 78.54 mm2 and thickness of 1 mm. U251 MG cells were seeded at a concentration of 1 3 106 cells/mL.ECT treatments with NaCl solutions are not shown due to no lesion being present. (***p< 0.0001).

(a)(b)

FIGURE 4. The numerical model of the experimental setup is used to determine the area of collagen exposed to various electricfield thresholds for applied voltages of 450 and 800 V. (a) Numerically integrating the finite element model of the collagen scaffoldfor different electric field thresholds allowed us to construct a plot that related the two variables. (b) Overlaying the electric fieldcontour lines taken from our numerical model with an image of a 5 mM CaCl2 treatment demonstrated the accuracy of the finiteelement model (scale bar 1 mm). The scaffold has a total area of 78.54 mm2.

The Feasibility of Enhancing Susceptibility of Glioblastoma Cells

voltage of 450 V was chosen because it provides a widerange of electric field magnitudes over a broad range ofareas. Figure 4b shows electric field contours takenfrom the numerical model of the collagen scaffold,overlaid onto a treatment using IRE in combinationwith 5 mM CaCl2 solution. Electric field lines of highmagnitude are plotted to locate the electrodes in theimage and place the overlaying solution accurately.The average field threshold using least squares fittingwas calculated to be 377 ± 18.6 V/cm. An electric fieldcontour of 400 V/cm demonstrates the accuracy of thenumerical solution in predicting cell death thresholds.

Figure 5 shows the electric field thresholds that werecalculated using the polynomial equation. IRE treat-ments used in combination with CaCl2 solutions hadsignificantly lower field thresholds than their NaClcontrols. A treatment using IRE and 1 mM CaCl2resulted in a field threshold of 467 ± 67 V/cm whereasIRE used in combination with 1 mM NaCl resulted ina threshold of 698 ± 103 V/cm. Increasing the amountof calcium in our solution to a 5 mM concentrationresulted in a further reduced threshold of 377 ± 19 V/cm. This threshold was significantly lower than itscontrol using 5 mM NaCl (745 ± 139 V/cm). IREtreatments using 5 mM CaCl2 resulted in a lowerthreshold than ECT treatments using 5 mM CaCl2(440 ± 26 V/cm), but the difference was not statisti-cally significant (p = 0.3853). The same is true for aCaCl2 concentration of 1 mM, which resulted in anaverage threshold value for an ECT treatment of481 ± 38 V/cm (p = 0.9997).

Simulating Lesion Volumes for an In Vivo Treatmentin the Brain

Calculating the field threshold for an in vitro cal-cium IRE treatment enabled us to estimate what willoccur during an in vivo treatment. Figure 6 shows theresults of our numerical simulation of ECT and IREcalcium treatments in a 3D model of the brain. Here,we have accounted for dynamic changes in conduc-tivity of the tissue due to electroporation and changesin temperature that result due to Joule heating.Figures 6a and 6d indicate the electric field distribu-tions in the tissue after an IRE and ECT treatmentrespectively along a cut plane in the xz axis. For anECT treatment, the electric field magnitude betweenthe electrodes does not reach as high a value as an IREtreatment. This is due to the low number of pulsesapplied (8 instead of 80).5 ECT pulses are designed toreversibly electroporate tissue and therefore themajority of the tissue does not reach a large enoughmagnitude to kill cells. The reversible regime of elec-troporation also covers a smaller area than an IREtreatment does.

Figure 6b shows the ablation zone for an IREtreatment without CaCl2 (5 mM NaCl control). Theablation zone is greatly increased when 5 mM CaCl2 isadded (Fig. 6c). An ECT treatment on the other hand,only has an ablation when CaCl2 is used (Fig. 6e).Comparing ECT and IRE ablations when 5 mM CaCl2is used (Fig. 6f) shows that the calcium IRE lesion islarger. These results are also reflected in Table 3.

******

FIGURE 5. Calculated electric field thresholds for collagen scaffold experiments demonstrate that CaCl2 treatments lead tosignificantly lower cell death thresholds than their NaCl controls. 3D collagen experiments consisted of 4.0 mm electrode spacing,100 ls pulse width, 1 Hz frequency and 450 V. For IRE, 80 pulses were delivered while for ECT, 8 pulses were delivered. Collagenscaffolds had a total area of 78.54 mm2 and thickness of 1 mm. U251 MG cells were seeded at a concentration of 1 3 106 cells/mL.ECT treatments with NaCl solutions are not shown due to no lesion being present. (***p< 0.0001).

WASSON et al.

Table 3 shows lesion volumes calculated using thenumerical model of the brain for applied voltages of2000 and 3000 V. For 3000 V, the lesion volume for a1 mM CaCl2 treatment using IRE pulses is 2.109larger than lesion size for a 1 mMNaCl treatment. Theincrease is even more substantial for a 5 mM CaCl2treatment using IRE pulses. At an average lesion vol-ume of 29.80 ± 1.57 cm3, this extends well beyond thebulk tumor margin.

Figure 7 reveals the relationship between appliedvoltage and lesion volume for the 3D model of thebrain. As voltage increases with constant electrode

spacing, the difference between a 5 mM CaCl2 tream-tent using ECT and IRE pulses grows substantially.When used in combination with IRE pulses, a concen-tration of 5 mM CaCl2 results in an increase in lesionvolume 1.49 an ECT treatment using the same con-centration of calcium solution. This drastic increase canbe attributed to the fact that both the irreversibly andreversibly electroporated regions are growing as thenumber of pulses delivered increases. The degree towhich the tissue is electroporated increaes dramaticallywith increased voltage, highlighting our ability tomodulate pulse parameters to optimize treatment.

FIGURE 6. Our numerical model simulating an in vivo treatment indicates IRE has a larger increase in lesion size than ECT whenused in combination with 5 mM CaCl2 (xz plane). (a) Electric field distribution (V/cm) after IRE treatment (3000 V, 2.0 cm electrodespacing, 80 pulses). (b) Temperature distribution after IRE treatment (�C); (c) Lesion size after IRE with 5 mM NaCl control (white);(d) Comparing lesion size for IRE with 5 mM NaCl control (white) and 5 mM CaCl2 (gray). (e) Electric field distribution (V/cm) afterECT treatment (3000 V, 2.0 cm electrode spacing, 8 pulses). (f) Temperature distribution after ECT treatment (�C) (g) Lesion sizeafter ECT treatment with 5 mM CaCl2. (h) Comparing lesion size for ECT (white) and IRE (gray) treatments in combination with 5 mMCaCl2.

TABLE 3. Simulated lesion volumes for each experimental condition calculated using a numerical model of the brain withdifferent applied voltages (electrode spacing of 2.0 cm).

Pulse Treatment Volume at 2000 V (cm3) Volume at 3000 V (cm3)

IRE 1 mM CaCl2 11.54 ± 2.96 23.56 ± 3.83

1 mM NaCl 2.77 ± 1.50 11.20 ± 4.69

5 mM CaCl2 15.30 ± 0.88 29.80 ± 1.57

5 mM NaCl 2.35 ± 1.93 9.34 ± 5.09

ECT 1 mM CaCl2 10.17 ± 1.40 18.90 ± 1.82

1 mM NaCl No lesion No lesion

5 mM CaCl2 11.68 ± 0.91 20.93 ± 1.42

5 mM NaCl No lesion No lesion

The Feasibility of Enhancing Susceptibility of Glioblastoma Cells

DISCUSSION

The overall goal of our study was to determine ifusing calcium in combination with IRE resulted inlarger lesions than IRE alone and ECT pulses com-bined with calcium. During an IRE treatment, therewill be four zones of electroporation: (1) small zone ofcell death caused by thermal damage (Joule heating),(2) medium sized zone of necrotic tissue in which cellsare electroporated, lose homeostasis and are unable torecover, (3) large zone of apoptotic cell death in whichdefects in the membrane close, but cells are unable torecover from a loss of homeostasis and (4) reversiblyelectroporated cells that recover and survive.27 Use ofcalcium IRE intensifies this cell death phenomenonand takes advantage of the reversibly electroporatedzone of cells by driving them to cell death using cal-cium, therefore enhancing our zone of ablation.

Lee et al., have discussed the role that calcium canplay in cell death after electroporation.27 This wasevidenced when cells were electroporated with a450 ms pulse with 150 V/cm magnitude and a sharpincrease in intracellular calcium levels was observed.Instead of a rapid drop in calcium concentration afterthe pulse was removed, the authors saw sustainedelevated levels for 10 min. A sudden influx of calciumions may lead cells to consume their supply of ATP topump excess calcium across the cell membrane. Thisdepletion of ATP after calcium electroporation hasbeen shown by Frandsen et al., after electroporatingChinese hamster lung fibroblast cells with ECT pulsesand 1 mM calcium.14 Intracellular ATP levelsdecreased markedly and remained at 10.3% of control

values eight hours after treatment. Furthermore, evi-dence has shown that elevated levels of intracellularcalcium may activate proteases and phospholipasesthat further damage the cell and membrane, preventingpores from resealing after electroporation.20

The possibility of enhancing ablation size by usingcalcium to drive cells in the reversibly electroporatedzone to undergo cell death is supported by our exper-imental results and numerical modeling. In Fig. 2,ECT treatments using NaCl solutions resulted in nocell death. Cells exposed to this ECT treatment arereversibly electroporated and are therefore able to re-cover and maintain viability. As evidenced by bothECT and sham treatments, the saline solution does notlead to cell death. Scaffolds exposed to IRE pulses inNaCl solutions, on the other hand, form a small regionof cell death extending beyond the immediate vicinityof the electrodes (13.2 ± 3.6 mm2 for 5 mM NaCl). Itcan be concluded that these cells experience a loss ofhomeostasis due to electroporation resulting in celldeath (zone 2 described above). For both ECT andIRE treatments using CaCl2, a large zone of cell deathformed in the scaffold. Since a comparable lesion wasnot observed for NaCl treatments using the sameapplied pulses, it can be concluded that the region ofcell death extending beyond what was seen with NaClis due to the action of calcium and not the buffer oradditional heating effects due to changes in conduc-tivity of the scaffold. A 1 mM CaCl2 treatment resultsin a lesion area of 25.1 ± 3.9 mm2 for IRE and23.8 ± 2.5 mm2 for ECT. A 5 mM CaCl2 treatmentresulted in much larger lesion areas for both IRE(32.5 ± 2.0 mm2) and ECT (26.7 ± 2.1 mm2). The factthat 1 mM CaCl2 results in a smaller lesion area than5 mM CaCl2 suggests that some cells in the peripheryof the reversibly electroporated zone can recover fromtreatment. Using a higher concentration (5 mM CaCl2)further exacerbates the effects of calcium, renderingthe cells unable to recover from the treatment, causingadditional cell death. The lesion area of our treatmentsmay be influenced by the lower conductivity of ourmedium, as shown previously by Pucihar et al.33 Ourlesion area results are consistent with the findings ofFrandsen et al., when comparing calcium ECT treat-ment to NaCl controls in vitro14 using three cell lines.The half maximal effective concentration of calcium(EC50) was found to be 0.57 mM. At 1 mM calcium,viability for all three cell lines ranged from 10–30%and at 3 mM calcium, viability for all three cell lineswas below 20%. Cell death saturated at 5 mM calciumwith viability for all three cell lines being below 10%.We have extended the implications of that study toprovide further evidence that calcium can also be usedto enhance lesion boundaries when used in combina-tion with IRE pulses.

FIGURE 7. Numerical modeling predicts larger lesion vol-ume in vivo for an IRE calcium treatment when compared toan ECT calcium treatment or IRE alone. The model consistedof two electrodes spaced 2.0 cm apart in a 3D domain of braintissue. Three different applied voltages were tested (1000,2000, 3000 V).

WASSON et al.

Calcium treatments decreased the required electricfield threshold for cell death. When treating with IREalone, we obtained a field threshold of 745 ± 139 V/cm whereas for 5 mM CaCl2, we obtained 377 ± 19 V/cm, just over half of its control. This result is similar tofindings of Hansen et al., where SW780 human bladdercancer cell lines were treated using ECT pulses withvarying applied electric field magnitudes.22 This sug-gests that it may be possible to increase lesion sizewithout increasing the applied voltage, mitigatingconcern of additional thermal damage with higherenergy IRE treatment.

Our numerical model that simulates a treatment inthe brain, predicts an efficacy enhancement for IREand calcium therapy that may be expected in a clini-cally relevant in vivo setting. A 5 mM CaCl2 IREtreatment resulted in an increase in lesion volumethree times that of its NaCl control. Comparable re-sults were found in a mouse model, when tumorstreated with 168 mM CaCl2 and electroporationdramatically decreased in volume immediately aftertreatment and continued to decrease in size up to28 days after treatment.14 We have previouslydemonstrated the safety and efficacy of IRE treat-ment for brain cancer in canine patients that pre-sented with spontaneous gliomas.12,15,17 Rossmeislet al., demonstrated that IRE did not lead to neuro-toxicity in six out of seven canine patients, increasingKarnofsky Performance Scale in all patients.34 It hasalso been previously reported that non-thermal IREtreatment spares critical structures such asnerves,28,32,37 therefore we do not have reason to be-lieve that neuron function will be impaired followingIRE treatment. In future studies, it will be necessaryto better characterize the effect of treatment on neu-rons and the increase in conductivity due to electro-poration in human brain tissue. Treatments will alsoneed to be simulated using 3D models reconstructedfrom patient MR images in order to ensure maximumaccuracy and efficacy.

It should be noted that despite most tissues havingextracellular calcium concentrations around 1 mM,calcium ions are often bound by other macromoleculesand only a small fraction are free in the extracellularfluid. Therefore, when treating tumors in vivo usingcalcium electroporation, administration of exogenouscalcium will be required for effect. Ensuring uniformdistribution of calcium in the tissue to be treated maybe difficult to achieve due to leaky vasculature, highinterstitial pressure and convective forces that drivefluid out of the tumor. The potential challenges asso-ciated with delivery of calcium needs to be investigatedin future research. Previously, Frandsen et al., hasdemonstrated that direct tumor injection in combina-tion with ECT has been successful in treating mice that

were injected with human small cell lung cancer cells.14

CaCl2 solutions were injected into the tumor at avolume that was 50% of tumor volume and the needlewas moved around the tumor to ensure uniforminjection. Using this technique, 89% of treated tumors(~1 cm3) were eliminated. It may also be possible tohave co-delivery of the treatment with a needle/elec-trode system, although this may present a challenge inand of itself and will be left for future work.

Calcium IRE may provide an advantage over cal-cium ECT for in vivo treatment of tumors since IREtreatment ensures that the bulk of the tumor will bekilled whereas ECT does not. While the bulk tumor isdestroyed, the reversibly electroporated regionextending beyond the tumor margin will experience aninflux of calcium, exacerbating cell death andenhancing the ablation margin.

CONCLUSION

We have demonstrated that CaCl2 treatment used incombination with IRE therapy leads to larger lesionsthan IRE alone. Consequently, the electric fieldthreshold needed to kill the cancer cells is reduced bynearly half, suggesting that larger lesions are attainablewithout an increase in applied energy. Results fromour numerical models indicate calcium IRE treatmentin vivo can lead to treatment volumes that are 1.49larger than calcium ECT treatments and 3.29 largerthan IRE alone. While this study focused on calciumIRE treatment of brain cancer cells, the implications ofthese results are applicable to many types of cancerthat are unresectable and invasive. Since calcium buf-fer solutions are regularly used in clinical settings andare known to be nontoxic to cells with intact cellmembranes, calcium IRE may provide a safe andeffective way to increase treatment volumes withoutinducing additional thermal damage.

ELECTRONIC SUPPLEMENTARY MATERIAL

The online version of this article (doi:10.1007/s10439-017-1905-6) contains supplementarymaterial, which is available to authorized users.

ACKNOWLEDGMENTS

This work was supported by the NSF GraduateResearch Fellowship Program under Grant No. DGE-1651272, the National Cancer Institute of the NationalInstitutes of Health under Award R01CA213423, theInstitute for Critical Technology and Applied Science

The Feasibility of Enhancing Susceptibility of Glioblastoma Cells

(ICTAS) of Virginia Tech, Wake Forest Comprehen-sive Cancer Center and NSF CAREER AwardsCBET-1055913 and CBET-1652112.

REFERENCES

1Agerholm-Larsen, B., H. K. Iversen, P. Ibsen, J. M.Moller, F. Mahmood, K. S. Jensen, J. Gehl, K. SvarreJensen, and J. Gehl. Preclinical validation of elec-trochemotherapy as an effective treatment for braintumors. Cancer Res. 71:3753–3762, 2011.2Al-Sakere, B., F. Andre, C. Bernat, E. Connault, P. Opo-lon, R. V. Davalos, B. Rubinsky, and L. M. Mir. Tumorablation with irreversible electroporation. PLoS ONE 2:1–8, 2007.3Arena, C. B., C. S. Szot, P. A. Garcia, M. N. Rylander,and R. V. Davalos. A three-dimensional in vitro tumorplatform for modeling therapeutic irreversible electropo-ration. Biophys. J. 103:2033–2042, 2012.4Bergman, T. L., A. S. Lavine, F. P. Incropera, and D. P.Dewitt. Fundamentals of Heat and Mass Transfer (7thed.). Hoboken: Wiley, 2011.5Bhatt, D. L., D. C. Gaylor, and R. C. Lee. Rhabdomyol-ysis due to pulsed electric fields. Plast. Reconstr. Surg.86:1–11, 1990.6Bier, M., S. M. Hammer, D. J. Canaday, and R. C. Lee.Kinetics of sealing for transient electropores in isolatedmammalian skeletal muscle cells. Bioelectromagnetics20:194–201, 1999.7Bonakdar, M., E. M. Wasson, Y. W. Lee, and R. V.Davalos. Electroporation of brain endothelial cells on chiptoward permeabilizing the blood-brain barrier. Biophys. J.110:503–513, 2016.8Chiche, J., M. C. Brahimi-Horn, and J. Pouyssegur. Tu-mour hypoxia induces a metabolic shift causing acidosis: acommon feature in cancer. J. Cell. Mol. Med. 14:771–794,2010.9Davalos, R. V., B. Rubinsky, and L. M. Mir. Theoreticalanalysis of the thermal effects during in vivo tissue elec-troporation. Bioelectrochemistry 61:99–107, 2003.

10Duck, F. A. Physical Properties of Tissue: A Comprehen-sive Reference Book. London: Academic Press, 1990.

11Edd, J. F., L. Horowitz, R. V. Davalos, L. M. Mir, and B.Rubinsky. In vivo results of a new focal tissue ablationtechnique: irreversible electroporation. IEEE Trans.Biomed. Eng. 53:1409–1415, 2006.

12Ellis, T. L., P. A. Garcia, J. H. Rossmeisl, N. Henao-Guer-rero, J. Robertson, and R. V. Davalos. Nonthermal irre-versible electroporation for intracranial surgical applications.Laboratory investigation. J. Neurosurg. 114:681–688, 2011.

13Fischbach, C., R. Chen, T. Matsumoto, T. Schmelzle, J. S.Brugge, P. J. Polverini, and D. J. Mooney. Engineeringtumors with 3D scaffolds. Nat. Methods 4:855–860, 2007.

14Frandsen, S. K., H. Gissel, P. Hojman, T. Tramm, J.Eriksen, and J. Gehl. Direct therapeutic applications ofcalcium electroporation to effectively induce tumor necro-sis. Cancer Res. 72:1336–1341, 2012.

15Garcia, P. A., R. E. Neal, J. H. Rossmeisl, and R. V.Davalos. Non-thermal irreversible electroporation for deepintracranial disorders. Conf. Proc. IEEE Eng. Med. Biol.Soc. 2743–2746, 2010.

16Garcia, P. A., J. H. Rossmeisl, R. E. Neal, T. L. Ellis, andR. V. Davalos. A parametric study delineating irreversibleelectroporation from thermal damage based on a minimallyinvasive intracranial procedure. Biomed. Eng. Online 10:34,2011.

17Garcia, P. A., J. H. Rossmeisl, R. E. Neal, T. L. Ellis, J. D.Olson, N. Henao-Guerrero, J. Robertson, and R. V.Davalos. Intracranial nonthermal irreversible electropora-tion: in vivo analysis. J. Membr. Biol. 236:127–136, 2010.

18Gehl, J. Electroporation: theory and methods, perspectivesfor drug delivery, gene therapy and research. Acta Physiol.Scand. 177:437–447, 2003.

19Gerweck, L. E., and K. Seetharaman. Cellular pH gradientin tumor versus normal tissue: potential exploitation for thetreatment of cancer. Cancer Res. 56:1194–1198, 1996.

20Gissel, H., and T. Clausen. Ca2+ uptake and cellular in-tegrity in rat EDL muscle exposed to electrostimulation,electroporation, or A23187. Am. J. Physiol.-Regul. Integr.Comp. Physiol. 285:R132–R142, 2003.

21Gothelf, A., L. M. Mir, and J. Gehl. Electrochemotherapy:results of cancer treatment using enhanced delivery ofbleomycin by electroporation. Cancer Treat. Rev. 29:371–387, 2003.

22Hansen, E. L., E. B. Sozer, S. Romeo, S. K. Frandsen, P. T.Vernier, and J. Gehl. Dose-dependent ATP depletion andcancer cell death following calcium electroporation, relativeeffect of calcium concentration and electric field strength.PLoS ONE 10:e0122973, 2015.

23Hjelmeland, A. B., Q. Wu, J. M. Heddleston, G. S.Choudhary, J. MacSwords, J. D. Lathia, R. McLendon, D.Lindner, A. Sloan, and J. N. Rich. Acidic stress promotes aglioma stem cell phenotype. Cell Death Differ. 18:829–840,2011.

24Ivey, J. W., E. L. Latouche, M. B. Sano, J. H. Rossmeisl,R. V. Davalos, and S. S. Verbridge. Targeted cellularablation based on the morphology of malignant cells. Sci.Rep. 5:17157, 2015.

25Ivorra, A., B. Al-Sakere, B. Rubinsky, and L. M. Mir. Invivo electrical conductivity measurements during and aftertumor electroporation: conductivity changes reflect thetreatment outcome. Phys. Med. Biol. 54:5949–5963, 2009.

26Lackovic, I., R. Magjarevic, and D. Miklavcic. Three-di-mensional finite-element analysis of joule heating in elec-trochemotherapy and in vivo gene electrotransfer. IEEETrans. Dielectr. Electr. Insul. 16:1338–1347, 2009.

27Lee, R. C., D. J. Canaday, and S. M. Hammer. Transientand stable ionic permeabilization of isolated skeletal musclecells after electrical shock. J. Burn Care Rehabil. 14:528–540, 1993.

28Li, W., Q. Fan, Z. Ji, X. Qiu, and Z. Li. The effects ofirreversible electroporation (IRE) on nerves. PLoS ONE6:e18831, 2011.

29Linnert, M., and J. Gehl. Bleomycin treatment of braintumors: an evaluation. Anticancer Drugs 20:157–164, 2009.

30Miklavcic, D., D. Semrov, H. Mekid, and L. M. Mir. Avalidated model of in vivo electric field distribution in tis-sues for electrochemotherapy and for DNA electrotransferfor gene therapy. Biochim. Biophys. Acta 1523:73–83, 2000.

31Neal, R. E., J. H. Rossmeisl, V. D’Alfonso, J. L. Robert-son, P. A. Garcia, S. Elankumaran, and R. V. Davalos.In vitro and numerical support for combinatorial irre-versible electroporation and electrochemotherapy gliomatreatment. Ann. Biomed. Eng. 42:475–487, 2014.

32Neal, R. E., J. H. Rossmeisl, P. A. Garcia, O. I. Lanz, N.Henao-Guerrero, and R. V. Davalos. Successful treatment

WASSON et al.

of a large soft tissue sarcoma with irreversible electropo-ration. J. Clin. Oncol. 29:e372–e377, 2011.

33Pucihar, G., T. Kotnik, M. Kanduser, and D. Miklavcic.The influence of medium conductivity on electroperme-abilization and survival of cells in vitro. Bioelectrochemistry54:107–115, 2001.

34Rossmeisl, J. H., P. A. Garcia, T. E. Pancotto, J. L.Robertson, N. Henao-Guerrero, R. E. Neal, T. L. Ellis,and R. V. Davalos. Safety and feasibility of the NanoKnifesystem for irreversible electroporation ablative treatment ofcanine spontaneous intracranial gliomas. J. Neurosurg.123:1008–1025, 2015.

35Rubinsky, B. Irreversible electroporation in medicine.Technol. Cancer Res. Treat. 6:255–259, 2007.

36Salford, L. G., B. R. R. Persson, A. Brun, C. P. Ceberg, P.C. Kongstad, and L. M. Mir. A new brain tumor therapycombining bleomycin with in vivo electropermeabilization.Biochem. Biophys. Res. Commun. 194:938–943, 1993.

37Schoellnast, H., S. Monette, P. C. Ezell, A. Deodhar, M.Maybody, J. P. Erinjeri, M. D. Stubblefield, G. W. Single,W. C. Hamilton, and S. B. Solomon. Acute and subacuteeffects of irreversible electroporation on nerves: experi-mental study in a pig model. Radiology 260:421–427, 2011.

38Sel, D., D. Cukjati, D. Batiuskaite, T. Slivnik, L. Mir, andD. Miklavcic. Sequential finite element model of tissueelectropermeabilisation. Conf. Proc. IEEE Eng. Med. Biol.Soc. 5:3551–3554, 2004.

39Stewart, L. A. Chemotherapy in adult high-grade glioma: asystematic review and meta-analysis of individual patientdata from 12 randomised trials. Lancet 359:1011–1018,2002.

40Stupp, R., W. Mason, M. J. van den Bent, M. Weller, B.M. Fisher, M. J. B. Taphoorn, K. Belanger, A. A.Brandes, C. Marosi, U. Bogdahn, J. Curschmann, R. C.Janzer, S. K. Ludwin, T. Gorlia, A. Allgeier, D. Lacombe,G. Cairncross, E. Eisenhauer, and R. O. Mirimanoff.Radiotherapy plus concomitant and adjuvant temozolo-mide for glioblastoma. N. Engl. J. Med. 352:987–996,2005.

41Uzuka, T., R. Tanaka, H. Takahashi, K. Kakinuma, J.Matsuda, and K. Kato. Planning of hyperthermic treat-ment for malignant glioma using computer simulation. Int.J. Hyperth. 17:114–122, 2001.

42Werner, J., and M. Buse. Temperature profiles with respectto inhomogeneity and geometry of the human body. J.Appl. Physiol. 65:1110–1118, 1988.

The Feasibility of Enhancing Susceptibility of Glioblastoma Cells