tu

rk, PriJ 0847d Stgy, A

anthederimt caconsistent with temperature measurements obtained from the experimental

of deatsecond only to cardiovascular diseases [1]. Bas

from cancer in 2015 and 11.4 million in 2030. At this rate, itmay surpass have shown that heat affects nuclear function through the inhibition

Materials Science and Engineering C 32 (2012) 22422249

Contents lists available at SciVerse ScienceDirect

Materials Science a

.e lcardiovascular disease as the leading cause of death by 2012 [2].Cancer is a generic term for a large group of diseases that can affect

any part of the body. Current scientic evidence suggests that cancercan be triggered by environmental and genetic factors [3]. Regardlessof the trigger, unless it is diagnosed in the early stages, the prognosisfor patients is often poor [46]. Current treatment modalities include:radiotherapy, chemotherapy, hormonal therapy and surgical removal[1]. When possible, surgical removal, in combination with other treat-ment modalities, often offers the best prognosis for patients [7].

However, common treatment modes such as radiotherapy andchemotherapy are known to induce multiple side effects that canhave long-lasting impact on a patient's quality of life [8] and hormonal

of RNA [1215], DNA [16,17], and protein synthesis [1618]. In addition,hyperthermia causes delay or arrest in cell cycle progression [19]; chief-ly through mitotic arrest [2023] and inhibiting S phase entry from G2[24,25]. Groups have also reported reduced cell metabolism followinghyperthermia [18,26,27]. Additionally, cells up-regulate heat shock pro-teins (HSPs) in response to heat treatment immediately following aprior heat treatment. This up-regulation of HSPs leads to a well-knownphenomenon, known as thermo-tolerance [11]. Consequently, ndingsfrom prior work justify investigating new pulsed heating regimentsthat would allow sufcient time for the thermo resistance to subsidewhile taking advantages of cell cycle disruptions [8,13,14].

In recent times, the rst ofcial clinical use of hyperthermia was in

therapy is only available to patients with cerConsequently, there has been increasing intea treatmentmodality because it has minimal

Corresponding author at: Princeton Institute for Scirials (PRISM), Princeton University, Princeton, NJ 0854258 5609; fax: +1 609 258 5877.

E-mail address: soboyejo@princeton.edu (W.O. Sobo

0928-4931/$ see front matter 2012 Elsevier B.V. Alldoi:10.1016/j.msec.2012.06.010ed on projections, cancerd 9 million people dying

treatment for cancer is based on its direct effect on cells. Heat causes acellular stress which triggers a cascade of molecular events. Studiesdeaths will continue to rise with an estimateHyperthermiaBreast cancer cellsCell cytoskeletonHeat shock proteinsLocalized cancer treatment

1. Introduction

Cancer is the second leading causeis shown to cause signicant physical changes in the cell cytoskeleton. Finally, the paper explores the effectsof hyperthermia on cell growth and cell death under isothermal and cyclic conditions. The underlying effectsof heat shock protein expression are elucidated before discussing the implications of the results for cancertreatment via localized hyperthermia.

2012 Elsevier B.V. All rights reserved.

h throughout the world;

synergistic effects when used in combination with radiotherapy andchemotherapy [911].

The biological rationale for the use of hyperthermia as a potentialKeywords: set-ups. The paper also examines the effects of isothermal heating on the cell morphology. Isothermal heatingAvailable online 16 June 2012temperature variations areAn in-vitro study of the effects of temperaand models

C. Theriault a,b, E. Paetzell a,b, R. Chandrasekar c, C. Baa Princeton Institute for Science and Technology of Materials (PRISM), Princeton Universityb Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, Nc Department of Electrical and Computer Engineering, Purdue University, West Lafayette, INd Department of Mechanical Engineering, University of Delaware, Newark, DE 19716, Unitee Department of Materials Science & Engineering, African University of Science & Technolo

a b s t r a c ta r t i c l e i n f o

Article history:Received 1 March 2011Received in revised form 30 April 2012Accepted 11 June 2012

This paper presents an implthermia. Heating, accompliswhich are tested under expto 45 C as studied on breas

j ourna l homepage: wwwtain types of cancer [9].rest in hyperthermia asside effects and potential

ence and Technology of Mate-4, United States. Tel.: +1 609

yejo).

rights reserved.re on breast cancer cells: Experiments

ey d, Y. Oni a,b, W.O. Soboyejo a,b,e,nceton, NJ 08544, United States544, United States907, United Statesatesbuja, Nigeria

able biomedical device for the localized killing of cancer cells through hyper-via resistive heating, is modeled using numerical heat transfer techniques,ental conditions. The effect of temperature in the therapeutic domain of 37ncer cell line MDA-MB-231 is also reported. The results show the predicted

nd Engineering C

sev ie r .com/ locate /msecthe early part of the 20th century, when it was used as a treatment forcervical cancer [11]. However, it was not until the 1970s that the mod-ern discipline of thermotherapy really emerged beyond the regime ofexperimentation [2830]. Nevertheless, due to limits in technologicaladvances, very few clinical studies were performed before the 1990s.However, by the turn of the 21st century, there was a renewed interestin hyperthermia research and clinical applications in local and regionalhyperthermia [3134].

Similar to other treatment modalities, hyperthermia's clinical ob-jective is to achieve the localized death of tumorigenic tissue withoutdamaging the surrounding normal tissue. In contrast to bulk systemictreatments, thermotherapy can be administered locally, regionally, orsystemically [11,3537]. In any case, it is possible to achieve a treat-ment that is locally administered at tumorigenic sites with minimaldamage to surrounding tissues.

Within the last decade, researchers have developed multiple deliverymodalities for hyperthermia and tested them in both invitro and invivoconditions as previously described [12,17,38,39]. The most common isthe use of ferromagnetic uids in combination with an oscillating mag-netic eld [4042]. Magnetic rods or needles have also been exploredas excitable sources of heat [43,44]. Other radio-frequency deliverymodalities that have beendeveloped includehigh intensity focusedultra-sound and high-frequency eddy currents. Furthermore, the use of intra-peritoneal surgeries as treatment modalities for both hyperthermia

applied modalities, the new device induces hyperthermia by the cyclicapplication of heat to an implantable device. Short heat pulses (15 min

2.2. Design and testing of a biocompatible implant for inducing hyperthermiausing resistive heating

The hyperthermia implant was designed to have two largesurfaces that are implanted perpendicular to the tumor growth axis.A 10102 mm geometry was used in order to efciently distributethe resistive heating element within the implant (Fig. 1). When con-nected to a standard AA battery and in dry air conditions, the implant'stemperature increased from room temperature (23 C) to 125 C in50 s.

To simulate the potential effect of in-vivo implantation, a similartest was run in a 50 mL test tube lled with 10 mL of aqueous solu-tion. The test tube was immersed in a water bath at 37 C and allowedto equilibrate before turning on the hyperthermia inducing device.The results showed that it took an average of 4 min for the device toincrease the temperature at the interface of the water bath and thesample to 41 C under static conditions (Fig. 2).

cell growth, leading to cell cycle arrest and eventually inducing cell

2243C. Theriault et al. / Materials Science and Engineering C 32 (2012) 22422249in duration) at 45 C for 45 min are followed by a 12 h relaxation period.The in-vitro responses of breast cancer cells are examined and the mea-sured temperatures are compared with predictions from a nite differ-ence model. The implications of the results are discussed along withsuggestions for future work.

2. Device design and fabrication

2.1. Device fabrication

The device that was used to apply heat shock treatments to thecell samples consisted of 100 m thick insulated copper wire embed-ded in PDMS (Elastomer Sylgard 184 from Corning with 1:10 curingagent:PDMS mass ratio). Approximately 0.8 m of wire was wrappedaround a PDMS block with dimensions 110.2 cm. The woundblock was then embedded in a PDMS package that was cured at80 C for 2.5 h. The wire was wound uniformly within the PDMS, asshown schematically in Fig. 1.

Fig. 1. Schematic of the hyperthermia inducing device. The hyperthermia inducing de-vice was fabricated out of isolated copper wire (d=0.2 mm) and PDMS. The copperwire was rst wound to cover an area of 1 cm2 and then placed in a mold. Freshly pre-pared PDMS was then poured into the mold and allowed to cure for 2.5 h at 80 C. Onceand/or high-dose chemotherapy is becomingmore prevalent in the treat-ment of cancers of the gastrointestinal tract and female or male internalreproductive organ cancers [45]. Apart from the peritoneal surgeries,there is a lack of post-treatment follow-ups and many physicians haveexpressed concerns about the long-lasting effects of hyperthermic-inducing modalities [5].

This paper presents the results of a combined experimental and com-putational study of the effects of temperature on the structure and deathof breast cancer cells. Heating is achieved via an implantable device thatuses resistive heating to induce cell death in the surrounding cells/tissue.The device was fabricated from polydimethylsiloxane(PDMS), an FDA-approved biocompatible polymer [46]. Unlike the current externally-un-molded, the device had the following dimensions: 110.2 cm.death after multiple cycles.

3. Experimental procedures

3.1. Cell culture experiments

All the cell culture experiments performed in this study were con-ducted on the breast cancer cell line MDA-MB-231 (ATCC, ManassasVA). The cells were grown at 37 C in a humidied environment withatmospheric CO2 levels. They were grown in Leibovitz's L-15 mediumsupplemented with 10% fetal bovine serum and 2% penicillin. The cellswere seeded for 12 h prior to the rst heat shock treatmentwith an ini-tial conuence of ~50%, resulting in a ~70% conuence sample at thestart of cyclic treatment. For all cell counting samples, the sampleswere counted and then seeded 12 h prior to heat shock treatment.

Fig. 2. Simulation of in-vivo implantation. Hyperthermia inducing device was im-mersed in 10 mL of DiH2O at 37 C and was connected to a 1.5 V AA battery for15 min. The average temperature of the 10 mL sample of DiH2O was recorded. Resultsshowed a rapid ramp-up followed by a period of equilibrium in the thermal diffusion.For the nal experimental conditions, a voltage of 1.5 V was appliedto the wire and the temperature within the samples was maintainedusing an Omega CN7533 Proportional Integral Differential (PID) con-troller. Temperature was measured using T thermocouples (Omega,Stamford, CT). In this way, hyperthermic heating was applied for45 min, followed by cooling to 37 C (relaxation) to simulate actual hy-perthermic cycles.

Since current hyperthermia regimens include heat exposure from45min to 2 h with relaxation periods lasting from 3 days to 2 weeks[3537], the pulsed-hyperthermia regimen presented here introducesthe idea of frequent short-duration heat exposures, more specicallypulse durations of 45 min followed by 12 h relaxation periods. The ra-tionale behind this new regimen system would be to induce multiplecytoskeletal reorganizations due to short heat pulses that should inhibitThe power to the device was turned off after 15 min.

2244 C. Theriault et al. / Materials Science and Engineering C 32 (2012) 224222493.2. Cell exposure to continuous hyperthermia

The effects of continuous hyperthermia treatments were assessedusing clonogenic and trypan blue exclusion assays. First, a trypan blueexclusion assay was used to analyze cellular growth under hyperther-mic conditions. Briey, multiple samples of MDA-MB-231 breast cancercell linewere cultured. Each sample set contained aminimumof four cellculture asks. Once the sample sets had reached ~70% conuence, theywere exposed to one of four experimental conditions for up to 72 h.The four experimental conditions were: (i) 37 C; (ii) 41 C; (iii) 43 C;and (iv) 45 C. The number of cells in each sample was counted at leastfour times using a hemocytometer and a trypan blue exclusion assay[47]. This was done at regular intervals of 24 h.

In parallel, clonogenic assays were conducted to assess the cellviability/colony-forming ability of the cells immediately after treatment[48]. MDA-MB-231 cells were grown at 37 C until they reached ~70%conuence. The cells were then placed under constant temperaturesof 37, 41, 43, or 45 C for up to 72 h. The cells were harvested immedi-ately after 24, 48 and 72 h (or 6 and 12 h for 45 C) of heat treatment,and counted using a hemocytometer. Subsequently, the cells were plat-ed into 6-well petri dishes, in order to plate 150 cells/well. They werethen allowed to grow into colonies for 710 days at 37 C, before xingthemwith a 6% glutaraldehyde and 0.5% crystal violet mixture. The col-onieswere then countedwith a colony counter pen. All the experimentswere performed in triplicates, and the results are reported as percent-ages of the colonies (% of control).

Insight into the effectiveness of continuous hyperthermia on cellkilling was given by the trypan blue exclusion assay, while cell repro-ductive death/viability was characterized with the clonogenic assays.Overall, trends in cell numbers were elucidated through both assays.

3.3. Cyclic heat shock procedure

A cyclic heat shock procedurewas used to stimulate the potential ef-fects of multiple heat shock therapy. Each cycle of treatment consistedof 45 min of exposure to hyperthermic temperatures with in- andbetween-cycle temperatures of 45 and 373 C, respectively. Thiswas followed by 12 h of incubation. During the heat exposure, a tem-perature gradient was achieved within the sample, with the center ofthe dish at 4548 C and the edge of the dish at 3941 C, as discussedabove. The experiments were carried out for 5, 10 and 20 cycles. Thecell populations were then analyzed using both hemocytometry andpropidium iodine staining.

3.4. Propidium iodine assay for both cell death and apoptotic bodyformation

Propidium iodine (PI) assayswere used to determine the amount andlocation of cell death within a sample. Cell death was assumed to be afunction of the local temperature, and thus a guide for the heat diffusion.PI, at a concentration of 1 mg/mL in L15mediumsupplementedwith 10%of FBS, was added directly to the samples, after a 12 h incubation periodfollowing the last heat treatment. Subsequently, the samples were incu-bated for 15 min, allowing for the PI to stain and label the nuclei of thedead cells. The cells were then xed with 3.7% formaldehyde for15 min. Finally, the samples were observed and imaged using a NikonEclipse 50i with a medium band blue excitation lter (Tokyo Japan).

3.5. Cell cytoskeleton imaging

The cells were grown on glass cover slips for 24 h and then exposedto heat shock in a circulating water bath for 15, 30, 45 and 60 min. Thesamples were then rinsed in PBS, and xed in 3.7% formaldehyde solu-tion in PBS for 15 min. After washing, the cells were stained and incu-bated with DRAQ5 (Biostatus Limited, Shepshed, Leicestershire, UK)

for nuclear staining for 5 min. The samples were then stained andincubated with Oregon Green 488 paclitaxel (Invitrogen, Carlsbad, CA)for tubulin labeling for 1 h. Subsequently, the cells were rinsed withPBS and labeled for actin with FITC-conjugated phalloidin (Sigma, St.Louis, MO) for 20 min. After several washes with PBS, the sampleswere mounted on slides using a mounting medium, Aqua Poly/Mount(Polysciences, Warrington, PA). A RS3 Spinning Disk Confocal micro-scope (Perkin Elmer, Waltham MA) with a 60 objective was thenused to examine the immuno-uorescence of the cytoskeleton proteins.

3.6. Effects of temperature (37 C, 41 C, 43 C, and 45 C) on heat shockprotein expression

To assess the effects of cyclic heating on heat shock protein expres-sion, 2.5 millionMDA-MB-231 cellswere cultured into 6-well plates at aconcentration of 0.5105 cells per mL. The cells were then grown for24 h at 37 C and fed fresh medium prior to heat shock treatment. Foreach heat shock treatment, cultured plate was heated at either 41 C,43 C or 45 C for 30 min. This was accomplished by direct immersionin a circulatingwater bath. The uniformbath temperatures serve as con-trol experiments to simulate the effects of constant temperatures. Thetemperatures in the medium were monitored with a PID controller. Inall cases, the culture plates reached the desired temperatures within5 min. A 6-well plate was kept in the incubator at 37 C to representthe control group. Medium was harvested from duplicate wells fromeach plate immediately after treatment and 12 h after treatment. Thisprocedurewas repeated every 12 h for 5 cycles to investigate the effectsof cyclic heating.

The number of cells was counted at each temperature to adjust theresults of the ELISA by the viable cell number. Two separate wellswere used exclusively for counting (to avoid collecting any viable cellsin the ELISA assay). These wells were harvested every 48 h and themean cell count and viability were determined by direct cell count ina hemocytometer using the trypan blue exclusion assay.

To determine the concentration of inducible HSP70 in cells, an ELISAassay was conducted following the manufacturer's instructions (R&DSystems, Minneapolis, MN). Briey, a standard curve was createdusing HSP70 standard solution. 100 L of standards and samples wasadded to a 96-well plate and incubated at room temperature for 2 h.Eachwell waswashedwith awash buffer three times and then incubat-ed with 100 L mAb against HSP70 at a concentration of 0.25 g/mL for2 h at room temperature. Wells were again washed three times, and100 L of Streptavidin-HRP (1:200 dilution from stock solution in buff-ered protein base) was added and incubated for 20 min at room tem-perature. Wells were washed three times and 100 L of substratesolution (1:1 ratio of hydrogen peroxide to tetramethylbenzidine)was added and incubated for 20 min at room temperature. Then, the re-action was stopped with 50 L of 2 N sulfuric acid, and the optical den-sity at 450 nm was measured using an ELISA plate reader.

3.7. Statistical analysis

All statistical analyses were performed using S-PLUS (Ver. 7.0,TIBCO Software Inc., Palo Alto, Ca). A one-sided student's t-test wasused to determine the statistical signicance between two samplesmeasuring the same variable. When comparing multiple treatmentswith multiple factors, a multiple sample analysis of variance (ANOVA)test was performed using Tukey's method. A p-value lower than 0.05was considered a signicant difference, and condence intervals weremade using an alpha of 0.05.

4. Modeling

4.1. Numerical modeling of heat transfer

A nite difference model was written in MATLAB (The MathWorks

Inc., Natick, MA) to calculate the heat diffusion prole from the device

to the cell culture medium. The model used Fourier's equation and anite difference scheme to predict the temperature gradient withina plane of the implant. Briey, a 25 cm2 square mesh was constructedand divided uniformly into 10,000 nodes. An initial condition of 37 Cwas imposed on the entire array at t=0. Four boundary conditionswere considered: two in the x, two in the y-axis.

The boundaries were the contour of the implants and of the sys-tem. By assuming that the distance to the boundary of the systemwas much larger than the size of the implant, heat diffusion in the fur-thest boundary region becomes negligible, allowing the coil boundarytemperatures to be set to 37 C. The steady-state temperature of eachnode was then calculated using central difference and forward differ-ence approximations of solutions to Fourier's laws.

4.2. Two-dimensional analysis of the heat diffusion from the hyperthermiabiocompatible device into a uid lled environment

This expression can be rearranged to solve for the temperature atnode Ti,j at the time n+1. This gives:

Tn1i;j Tni;j tTni1;j Tni1;j Tni;j1 Tni;j14Tni;j

2

!3

where =x=y for a square mesh.This second order partial differential equation in Eq. (3) requires

that there must be 4 boundary conditions (two in the x directionand two in the y direction) as well as 1 initial condition at t=0. Therst heat treatment for each sample was started after an incubationperiod of about 12 h, and therefore, the initial temperature of the en-tire sample can be set at 37 C. Assuming a small temperature gradi-ent between the implant and the initial temperature and largeimplant-to-boundary distance, all nodes on the outer edges of thegrid can also be set at an incubation temperature of 37 C.

5. Results

5.1. Isothermal hyperthermia

The results of the in-vitro continuous hyperthermia experimentsare presented in Fig. 4(a) and (b). Both gures show cell culturedata that was obtained fromMDA-MB-231 breast cancer cell incubat-ed for up to 72 h at constant hypothermic conditions. Fig. 4(a) showsthe growth rate of this particular cell line at 37 C, as well as the se-vere decrease in growth, as the temperature increased. Necrosis is ob-served above 43 C, which is similarly reported for other cell lines

-50%N

2245C. Theriault et al. / Materials Science and Engineering C 32 (2012) 22422249To determine the temperature gradient in the area surrounding theimplant, the region was rst divided into an evenly distributed squaremesh. The grid consisted of 100 nodes dened along both x and y-axes, for an overall distribution of 10,000 total nodes. Each node wasrepresented by an area of x by y, as shown in Fig. 3.

The implant with square cross-sectional area of 1 cm2 was consid-ered to occupy 2020 nodes in the center of the array. The nodes oc-cupied by the implant were given initial conditions ranging from 45to 55 C, while those outside of the system were set at 37 C. TheMATLAB programwas then used to move forward in time, performingiterations until steady-state temperature conditions were reached.

An energy balance was performed on each node using the nitedifference method (implementing the explicit method) in conjunc-tion with the linear heat diffusion equation. The derivation for eachtemperature equation is governed by a two-dimensional form ofFourier's law of conduction. This is given by:

d2Tdx2

d2T

dy2 1

kg 1

dTdt

1

where g is dened as the heat generation term, and k and are thethermal conductivity and thermal diffusivity of the medium, respec-tively. Neglecting the heat generation term and applying a forward-time, central difference discretization for an interior node with noheat generation yields:

Tni1;j2Tni;j Tni1;jx2

Tni;j12Tni;j Tni;j1

y2 1Tn1i;j Tni;j

t2

Fig. 3. Graphical representation of algorithm used for modeling temperature gradient.A square grid pattern was used to model the heat diffusion in static conditions. These

conditions were very similar to the actual in-vitro testing conditions.0

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Fig. 4. Effect of different temperatures of continuous hyperthermia on MDA-MB-231Cell Population. Breast cancer cells MDA-MB-231 were incubated continuously at ei-ther 37, 41, 43, or 45 C for 72 h or until the population collapsed. Results are shown[4952]. Treatment at 43 C is shown to be the optimal temperaturefor hyperthermia treatments, which is consistent with reports in theliterature [53]. Fig. 4(b) shows similar trends for colony growth.

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5.2. Comparison of measured and predicted temperatures proles ofdevice mediated hyperthermia

In-vitro experimental observation showed that steady-state tem-perature conditions were achieved in a very short period of time(less than 4 min). It was, therefore, determined that the transient re-sponse was not very signicant. Consequently, only the steady-statetemperature distributions were examined. Typical results from thetemperature modeling are presented in Fig. 5(a). These show thatthe temperatures decrease with increasing distance from the centerof the device. The modeling results were supported by the in-vitro ex-periments. These were found to be in close agreement with thepredicted temperatures from the MATLAB simulation, as shown inFig. 5(b).

5.3. Staining of cell death and cell morphology under isothermalhyperthermia

The MDA-MB-231 cells were cultured in 60 mm Petri dishes (BD,Franklin Lakes, NJ) for 12 h and then isothermally heated (~2 h) toevaluate the effects of the heat treatment on the cell populations.Using our implantable device, the center of the sample was broughtto 55 C, while the outside edge remained below 41 C. Visual inspec-tion of PI staining of these samples showed the presence of both ne-crotic and apoptotic cell death, with necrosis prevalent in the region

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2246 C. Theriault et al. / Materials Science and Engineering C 32 (2012) 22422249Fig. 5. Temperature variation radiating from hyperthermia implant. Results are shownfrom (a) modeling the thermal diffusion from the hyperthermia device into its sur-roundings using MATLAB. The implant is set at 55 C and the outside extremitieswere set to 37 C (scale: 10 unit=0.5 cm). The results from the model (blue) are com-10 20 30 40 50 60 70 80 90 100

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bpared with the average experimental measurements (red) in (b).of high-hyperthermia (>45 C) and the apoptotic cell death in thelower-temperature regime (Fig. 6). This result is consistent with pre-vious studies linking necrotic cell death with temperatures in theupper-hyperthermic range and apoptotic cell death with tempera-tures in the lower-hyperthermic range [11,52,54,55].

Confocal microscopy images of cell samples exposed to 15, 30, 45and 60 min of hyperthermia at 43 C are presented in Fig. 7. The sam-ples were xed and stained for cytoskeletal structures including actin,microtubules, and nucleus. No signicant difference was observed be-tween the cytoskeletons of the control and the samples exposed for15 min at 43 C. The most signicant changes were observed in themicrotubule network. Specically, after 30 min of exposure, changesin the microtubule networks were observed. Some condensation ofthe actin cytoskeleton was also observed. After 45 min of exposure,both the microtubules and the actin network appeared to be affected.Furthermore, aggregation of the microtubule network was observedat 45 and 60 min. Actin networks were more disorganized after60 min, compared to those that were observed after 45 min.

Nuclear changes were present in approximately one third of thesamples exposed for 60 min. For those that did exhibit some nuclearchanges, mitotic body formation could be observed; presumably indi-cating that these cells had entered apoptosis. These results clearlyshow that after 30 min of exposure to 43 C, the cells started to expe-rience signicant deterioration of their cytoskeleton, which contin-ued with increased heat exposure.

5.4. Cyclic hyperthermia

The results of the cyclic hyperthermia experiments are presented inFig. 8(a) and (b). There is a statistically signicant difference betweenthe control and hyperthermia treatment at all cycles (pb5105). Al-though there is a decline in cell population after 5 hyperthermia treat-ment cycles, statistical analysis revealed that it was not signicant. Infact, the analysis showed that there was no signicant difference be-tween the mean number of cells at the beginning of the experimentsand after 5 or 10 cycles. However, after 20 cycles, a statistically signi-cant decrease was observed in the cell populations, when comparedto the other treatment cycles.

5.5. In-vitro cyclic heating and heat shock protein expression

The PI staining showed clearly that cell death propagated radiallyoutwards from the device after cyclic hyperthermia (Fig. 8(b)). Simi-lar trends were observed in the cell death proles after isothermalheating at 43 C (Fig. 6). The most prominent form of cell deathseems to be apoptosis, as demonstrated by the numerous apoptotic-bodies present in the samples. These results also show that at highertemperatures, fewer cycles are needed to induce cell death. More-over, the results also suggest that cell-cycle arrest was achieved with-in a large range of temperatures, ranging from 41 to 47 C. The resultsshow that heat shock protein expression increases with increasingtemperature between 41 and 47 C (Fig. 9).

Prior work has shown that heat shock proteins are proteins that areover-expressed during hyperthermia. In most tumor cells, they are al-ready over-expressed when compared to the basal level in normal cells[52,5658]. Recent work suggests that HSPs have the ability to serve ascarriers of tumor antigens and inammatory agents. Specically, multi-ple studies have shown that HSP70 interacts with receptors on antigenpresenting cells and can mediate T-lymphocytes to trigger an immuneresponse against the cells that are expressing HSP70 [57,59,60]. Amajor determinant of immune response is the interaction between theantigen-presenting cells and the T lymphocytes [61] due to its vasodila-tation properties. The current results (Fig. 9) show that the extent of HSPexpression increases with increasing temperature. The above interac-tions are, therefore, likely to increase with increasing temperature, for

temperatures between 41 and 47 C.

6. Discussion

6.1. Morphological changes in the cell cytoskeleton as a potential cause

cycle arrest, MDA-MB-231 cells start to enter the apoptotic pathway,most likely as a direct consequence of the cell cycle arrest or of mitot-ic block. Although more work is needed, our immuno-urescence re-

Fig. 6. Propidium iodine staining of 2 h continuous temperature exposure with the hyperthermia device. MDA-MB-231 cells were exposed to propidium iodine (PI) and xed. DNA uo-rescent labelingwas assayedusing 488 nmexcitation light source. Shown above are images from three regions of interest: the center (A), theposition (B), and the edge (C). Scale bars inbottom right corner correspond to 100 m; positions A, B, and C were approximately 0.5, 2, and 4 cm from the edge of the hyperthermia device. A1, B1, and C1 show the xed sample asviewedunder regular lightmicroscopy. All three images show a similar cell distribution pattern, indicating a homogeneous cell distribution. A2, B2, and C2 show the PI stain indicating theamount of cells with a ruptured nuclear membrane in each region (red labeling). A clear radial pattern emerges from the pictures with higher concentration of stained nucleus, a markerfor cellular death, towards the middle of the sample.

2247C. Theriault et al. / Materials Science and Engineering C 32 (2012) 22422249for both the disturbance of cellular function and for the cell's thermo-sensitive properties

Prior research has shown that heat shocks can have negative ef-fects on the cell cycle, often causing a temporary arrest that can lastfrom 2 to 14 h [52,62]. Our results suggest that, after 5 days of cellFig. 7. Cytoskeleton of MDA-MB-231 cells exposed to hyperthermia for different amount of tand 60 min. No signicant differences could be observed in the samples that were exposedsignicant aggregation of the microtubule network could be observed which increased wit30 min of exposure. At 60 min no clear actin network organization could be observed in thabove) was only observed in about one third of cells after 60 min of exposure.sults do support the hypothesis of mitotic arrest as the primarytrigger for apoptosis in our cell populations (Fig. 7).

The above observations are consistent with recent publications[32]. These suggest that heat induces conformational changes in themicrotubules structures, or in another regulatory protein, hence in-terfering with normal polymerization mechanisms and blocking theime. Confocal microscopy was performed on samples exposed to 43 C for 0, 15, 30, 45,to 43 C for 15 min compared to the control. After 30 min of hyperthermia exposure,h continued exposure. Differences in the actin laments could be observed starting ate majority of samples. Nuclear deterioration in the form of mitotic bodies (as shown

2248 C. Theriault et al. / Materials Science and Engineering C 32 (2012) 22422249cell at the mitotic spindle assembly checkpoint. Recently, Michalakiset al. [63] showed very similar results pertaining to the microtubuleorganization of HeLa cells after 1 h exposure to a 39 C heat shock.Their research also showed similarity between cells treated withheat shock and tubulin inhibitor (paclitaxel, arsenide, etc), indicatingthat mitotic arrest is the most likely the major apoptosis pathway.

Fig. 8. Growth as a function of heat shock cycles. (a) MDA-MB-231 cell samples wereexposed to multiple cycles of a heat shock duration of 45 min followed by a relaxationperiod of 12 h. Cell growth arrest was observed for the rst 10 cycles with a small non-statistically signicant decrease in the cell population after 10 cycles. After 20 cycles ofheat shock treatment, a large statistically signicant drop (pb5105) in the cell pop-ulation was observed. The underlying cause of cell death was hypothesized to be theprolonged cell cycle arrest caused by the cyclic heat shock as 45 min of exposure to43 C was shown to be non-lethal on its own. (b) PI staining of MDA-MB-231 cellsshowed a radial pattern of cell death: (bA) shows the cell population and (bB) showsthe cells that have died in the population. The hyperthermia device is located at the ex-treme right of the gure. This result was similar to the pattern observed during contin-uous hyperthermia exposure. Scale bar in lower right corresponds to 500 m.

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ck P

rote

in (n

g per

106 c

ells

)

Heat Shock Trial (12hr Intervals)

37C

41C

43C

45C

Fig. 9. Heat shock protein assay results. The extent of HSP expression fromMDA-MB-231cells increases with increasing temperature.6.2. The biocompatible hyperthermia device and its potential for medicalapplications

Although it is clear that this device could be used for treating a widerange of cancers, thiswork suggests that initial attention could be paid tobreast cancer, specically in patients with stage II or III non-metastatictumors. In such scenarios, surgical removal of the tumor is normally pos-sible, and radiotherapy as well as low doses of chemotherapy are stillprescribed in an effort to quench the body of any remaining cancercells and improve the patient's prognosis. As an alternative, the hyper-thermia device developed in this study could be implanted during thesurgical removal of a tumor. The device could then not only serve as astand-alone treatment modality, but could be used in combinationwith either radiotherapy or chemotherapy. Moreover, multiple studies[34,64] have shown additive and even synergistic results when hyper-thermia is combined with radiotherapy.

Prior work on internal heat and pain receptors have also shown thatsub-dermal temperatures of up to 43 C are very well tolerated clinical-ly, with patients feeling only a slight discomfort [65]. Furthermore, theblood perfusion rate, convection due to surrounding arteries, and uni-form thermal values could be taken into account using the bio-heattransfer equations [66,67]. Under such conditions, the heat ow canbe described by a three-dimensional (3D) heat diffusion model ratherthan the two-dimensional version presented here. A 3D model could,therefore, be used to predict which regions of the surrounding tissuewill reach hyperthermic temperatures by taking into account the actualpatient-specic data retrieved from clinical imaging techniques, such asMRI and ultrasound.

6.3. The potential use of an inductively coupled system for powerautonomy

In order for a hyperthermia device to be clinically efcient, there isa need for it to achieve a certain level of power autonomy. One poten-tial design would be to create an implant consisting of a closed coilembedded in PDMS. By introducing an alternating magnetic eld(AMF) in its vicinity, a current could be induced, thereby enabling re-sistive heating. The surrounding temperature would also have to bemonitored and controlled using wireless technology to maintain hy-perthermic temperatures. One potential pitfall with this system in-cludes the fact that the implant coil would have to be oriented in aspecic direction within the body for greatest power efciency.

In any case, the resulting current ow could then be stored in a re-chargeable battery to provide an autonomous power source. Furtherwork is clearly needed to demonstrate the feasibility of such systems.These should be combinedwith efforts to develop autonomous powersupplies for sustained use in the human body. More advanced tem-perature controls are also warranted. Finally, future versions of thedevice should be capable of automatically delivering the right amountof heat at the correct intervals without human interaction.

6.4. Potential future directions

Future work is needed to determine the response of normal breastcells, as well as other cancer cell lines, to the effects of heat. A recentstudy by Rylander et al. [68] showed that the HSP expression prole ofcancerous prostate cells was more sensitive to hyperthermia than theirnon-cancerous counterparts. In future work, uorescence-activated cellsorting (FACS), using propidium iodine or Annexin V, could be used toexplore the portions of cells that are in early or late stage apoptosis, ornecrosis. Furthermore, FACS cell cycle analyses should reveal at whatpoint hyperthermia causes mitotic arrest. Additionally, further quanti-cation of the cellular HSP expression prole in relation to cellular prox-imity to the hyperthermia-inducing device could provide additional

insights.

Furthermore, since the cellular responses of 2D models have beenshown to be different from 3D in-vivo models, there is a need to ex-plore the response to heat in more complex 3D in-vitro or in-vivomodels that simulate tumor conditions. This could be done in 3Dmicro-environments or in-vivo models that could further investigatethe use of regular short hyperthermia pulses in the selective killingof cancer cells. Such experiments could be performed with our with-out the proposed hyperthermia delivery device.

Also, although the current results do indicate that, in a simple 2Dcell culture environment, a pulsed hyperthermia regimen was efca-

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2249C. Theriault et al. / Materials Science and Engineering C 32 (2012) 22422249micro-environments can be simulated using gels and agars to mimictissue-like structures. The relationships between cell viability and dis-tance from the device should also be modeled quantitatively [69]after heat exposures in such 3D micro-environments. These are clear-ly some of the challenges for future work.

7. Summary and concluding remarks

This paper describes the results of an experimental and computa-tional study of the effects of localized hyperthermia on breast cancercells. Controlled hyperthermia was achieved using an implantable de-vice. The results from the in-vitro studies show clearly that low levelsof hyperthermia, when delivered in a short pulse regimen, can causecell cycle arrest in tumor cells. This leads to the inhibition of cell growthand increasing cell death after more than ve days of cyclic hyperther-mia. The observed increase in cell death is also associated with heatshock protein expression. This study suggests that, after 5 days of cyclicheat treatment, using the implantable device, MDA-MB-231 cells un-dergo cell cycle arrest.

The current results clearly suggest a need for in-vivo testing. Further-more, there is a need for additional studies that use surface texture topromote the improved integration of the device with biological tissue.

Acknowledgments

This workwas supported by theNational Science Foundation (GrantNo. DMR 0231418), the Grand Challenges Program at Princeton Univer-sity and the sponsor-id="gs3" id="gts0015">STEP-B Program of theWorld Bank. The authors are grateful to these organizations for theirsupport of the research.

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for breast cancer, PhD Thesis, Ohio State University, 2008.cious in halting cellular growth in MDA-MB-231 cells, in-vitro 3D

An in-vitro study of the effects of temperature on breast cancer cells: Experiments and models1. Introduction2. Device design and fabrication2.1. Device fabrication2.2. Design and testing of a biocompatible implant for inducing hyperthermia using resistive heating

3. Experimental procedures3.1. Cell culture experiments3.2. Cell exposure to continuous hyperthermia3.3. Cyclic heat shock procedure3.4. Propidium iodine assay for both cell death and apoptotic body formation3.5. Cell cytoskeleton imaging3.6. Effects of temperature (37C, 41C, 43C, and 45C) on heat shock protein expression3.7. Statistical analysis

4. Modeling4.1. Numerical modeling of heat transfer4.2. Two-dimensional analysis of the heat diffusion from the hyperthermia biocompatible device into a fluid filled environment

5. Results5.1. Isothermal hyperthermia5.2. Comparison of measured and predicted temperatures profiles of device mediated hyperthermia5.3. Staining of cell death and cell morphology under isothermal hyperthermia5.4. Cyclic hyperthermia5.5. In-vitro cyclic heating and heat shock protein expression

6. Discussion6.1. Morphological changes in the cell cytoskeleton as a potential cause for both the disturbance of cellular function and ...6.2. The biocompatible hyperthermia device and its potential for medical applications6.3. The potential use of an inductively coupled system for power autonomy6.4. Potential future directions

7. Summary and concluding remarksAcknowledgmentsReferences