Materials Science and Engineering C 32 (2012) 22422249

Contents lists available at SciVerse ScienceDirect

Materials Science and Engineering C

journal homepage: www.elsevier.com/locate/msec

An in-vitro study of the effects of temperature on breast cancer cells: Experiments

and models
C. Theriault a, b, E. Paetzell a, b, R. Chandrasekar c, C. Barkey d, Y. Oni a, b, W.O. Soboyejo a, b, e,
a

Princeton Institute for Science and Technology of Materials (PRISM), Princeton University, Princeton, NJ 08544, United States
Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, NJ 08544, United States
Department of Electrical and Computer Engineering, Purdue University, West Lafayette, IN 47907, United States
d
Department of Mechanical Engineering, University of Delaware, Newark, DE 19716, United States
e
Department of Materials Science & Engineering, African University of Science & Technology, Abuja, Nigeria
b
c

a r t i c l e

i n f o

Article history:
Received 1 March 2011
Received in revised form 30 April 2012
Accepted 11 June 2012
Available online 16 June 2012
Keywords:
Hyperthermia
Breast cancer cells
Cell cytoskeleton
Heat shock proteins
Localized cancer treatment

a b s t r a c t
This paper presents an implantable biomedical device for the localized killing of cancer cells through hyperthermia. Heating, accomplished via resistive heating, is modeled using numerical heat transfer techniques,
which are tested under experimental conditions. The effect of temperature in the therapeutic domain of 37
to 45 C as studied on breast cancer cell line MDA-MB-231 is also reported. The results show the predicted
temperature variations are consistent with temperature measurements obtained from the experimental
set-ups. The paper also examines the effects of isothermal heating on the cell morphology. Isothermal heating
is shown to cause signicant physical changes in the cell cytoskeleton. Finally, the paper explores the effects
of hyperthermia on cell growth and cell death under isothermal and cyclic conditions. The underlying effects
of heat shock protein expression are elucidated before discussing the implications of the results for cancer
treatment via localized hyperthermia.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Cancer is the second leading cause of death throughout the world;
second only to cardiovascular diseases [1]. Based on projections, cancer
deaths will continue to rise with an estimated 9 million people dying
from cancer in 2015 and 11.4 million in 2030. At this rate, it may surpass
cardiovascular 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 cancer
can be triggered by environmental and genetic factors [3]. Regardless
of the trigger, unless it is diagnosed in the early stages, the prognosis
for 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 treatment modalities, often offers the best prognosis for patients [7].
However, common treatment modes such as radiotherapy and
chemotherapy are known to induce multiple side effects that can
have long-lasting impact on a patient's quality of life [8] and hormonal
therapy is only available to patients with certain types of cancer [9].
Consequently, there has been increasing interest in hyperthermia as
a treatment modality because it has minimal side effects and potential
Corresponding author at: Princeton Institute for Science and Technology of Materials (PRISM), Princeton University, Princeton, NJ 08544, United States. Tel.: + 1 609
258 5609; fax: + 1 609 258 5877.
E-mail address: soboyejo@princeton.edu (W.O. Soboyejo).
0928-4931/$ see front matter 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.msec.2012.06.010

synergistic effects when used in combination with radiotherapy and

chemotherapy [911].
The biological rationale for the use of hyperthermia as a potential
treatment for cancer is based on its direct effect on cells. Heat causes a
cellular stress which triggers a cascade of molecular events. Studies
have shown that heat affects nuclear function through the inhibition
of RNA [1215], DNA [16,17], and protein synthesis [1618]. In addition,
hyperthermia causes delay or arrest in cell cycle progression [19]; chiefly through mitotic arrest [2023] and inhibiting S phase entry from G2
[24,25]. Groups have also reported reduced cell metabolism following
hyperthermia [18,26,27]. Additionally, cells up-regulate heat shock proteins (HSPs) in response to heat treatment immediately following a
prior heat treatment. This up-regulation of HSPs leads to a well-known
phenomenon, known as thermo-tolerance [11]. Consequently, ndings
from prior work justify investigating new pulsed heating regiments
that would allow sufcient time for the thermo resistance to subside
while taking advantages of cell cycle disruptions [8,13,14].
In recent times, the rst ofcial clinical use of hyperthermia was in
the early part of the 20th century, when it was used as a treatment for
cervical cancer [11]. However, it was not until the 1970s that the modern discipline of thermotherapy really emerged beyond the regime of
experimentation [2830]. Nevertheless, due to limits in technological
advances, very few clinical studies were performed before the 1990s.
However, by the turn of the 21st century, there was a renewed interest
in hyperthermia research and clinical applications in local and regional
hyperthermia [3134].

C. Theriault et al. / Materials Science and Engineering C 32 (2012) 22422249

Similar to other treatment modalities, hyperthermia's clinical objective is to achieve the localized death of tumorigenic tissue without
damaging the surrounding normal tissue. In contrast to bulk systemic
treatments, thermotherapy can be administered locally, regionally, or
systemically [11,3537]. In any case, it is possible to achieve a treatment that is locally administered at tumorigenic sites with minimal
damage to surrounding tissues.
Within the last decade, researchers have developed multiple delivery
modalities for hyperthermia and tested them in both invitro and invivo
conditions as previously described [12,17,38,39]. The most common is
the use of ferromagnetic uids in combination with an oscillating magnetic eld [4042]. Magnetic rods or needles have also been explored
as excitable sources of heat [43,44]. Other radio-frequency delivery
modalities that have been developed include high intensity focused ultrasound and high-frequency eddy currents. Furthermore, the use of intraperitoneal surgeries as treatment modalities for both hyperthermia
and/or high-dose chemotherapy is becoming more prevalent in the treatment of cancers of the gastrointestinal tract and female or male internal
reproductive organ cancers [45]. Apart from the peritoneal surgeries,
there is a lack of post-treatment follow-ups and many physicians have
expressed concerns about the long-lasting effects of hyperthermicinducing modalities [5].
This paper presents the results of a combined experimental and computational study of the effects of temperature on the structure and death
of breast cancer cells. Heating is achieved via an implantable device that
uses resistive heating to induce cell death in the surrounding cells/tissue.
The device was fabricated from polydimethylsiloxane(PDMS), an FDAapproved biocompatible polymer [46]. Unlike the current externallyapplied modalities, the new device induces hyperthermia by the cyclic
application of heat to an implantable device. Short heat pulses (15 min
in 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 measured temperatures are compared with predictions from a nite difference model. The implications of the results are discussed along with
suggestions for future work.

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2.2. Design and testing of a biocompatible implant for inducing hyperthermia

using resistive heating
The hyperthermia implant was designed to have two large
surfaces that are implanted perpendicular to the tumor growth axis.
A 10 10 2 mm geometry was used in order to efciently distribute
the resistive heating element within the implant (Fig. 1). When connected to a standard AA battery and in dry air conditions, the implant's
temperature increased from room temperature (23 C) to 125 C in
50 s.
To simulate the potential effect of in-vivo implantation, a similar
test was run in a 50 mL test tube lled with 10 mL of aqueous solution. The test tube was immersed in a water bath at 37 C and allowed
to equilibrate before turning on the hyperthermia inducing device.
The results showed that it took an average of 4 min for the device to
increase the temperature at the interface of the water bath and the
sample to 41 C under static conditions (Fig. 2).
For the nal experimental conditions, a voltage of 1.5 V was applied
to the wire and the temperature within the samples was maintained
using an Omega CN7533 Proportional Integral Differential (PID) controller. Temperature was measured using T thermocouples (Omega,
Stamford, CT). In this way, hyperthermic heating was applied for
45 min, followed by cooling to 37 C (relaxation) to simulate actual hyperthermic cycles.
Since current hyperthermia regimens include heat exposure from
45 min to 2 h with relaxation periods lasting from 3 days to 2 weeks
[3537], the pulsed-hyperthermia regimen presented here introduces
the idea of frequent short-duration heat exposures, more specically
pulse durations of 45 min followed by 12 h relaxation periods. The rationale behind this new regimen system would be to induce multiple
cytoskeletal reorganizations due to short heat pulses that should inhibit
cell growth, leading to cell cycle arrest and eventually inducing cell
death after multiple cycles.

3. Experimental procedures
2. Device design and fabrication

3.1. Cell culture experiments

2.1. Device fabrication

All the cell culture experiments performed in this study were conducted on the breast cancer cell line MDA-MB-231 (ATCC, Manassas
VA). The cells were grown at 37 C in a humidied environment with
atmospheric CO2 levels. They were grown in Leibovitz's L-15 medium
supplemented with 10% fetal bovine serum and 2% penicillin. The cells
were seeded for 12 h prior to the rst heat shock treatment with an initial conuence of ~50%, resulting in a ~70% conuence sample at the
start of cyclic treatment. For all cell counting samples, the samples
were counted and then seeded 12 h prior to heat shock treatment.

The device that was used to apply heat shock treatments to the
cell samples consisted of 100 m thick insulated copper wire embedded in PDMS (Elastomer Sylgard 184 from Corning with 1:10 curing
agent:PDMS mass ratio). Approximately 0.8 m of wire was wrapped
around a PDMS block with dimensions 1 1 0.2 cm. The wound
block was then embedded in a PDMS package that was cured at
80 C for 2.5 h. The wire was wound uniformly within the PDMS, as
shown schematically in Fig. 1.

Fig. 1. Schematic of the hyperthermia inducing device. The hyperthermia inducing device was fabricated out of isolated copper wire (d = 0.2 mm) and PDMS. The copper
wire was rst wound to cover an area of 1 cm2 and then placed in a mold. Freshly prepared PDMS was then poured into the mold and allowed to cure for 2.5 h at 80 C. Once
un-molded, the device had the following dimensions: 1 1 0.2 cm.

Fig. 2. Simulation of in-vivo implantation. Hyperthermia inducing device was immersed in 10 mL of DiH2O at 37 C and was connected to a 1.5 V AA battery for
15 min. The average temperature of the 10 mL sample of DiH2O was recorded. Results
showed a rapid ramp-up followed by a period of equilibrium in the thermal diffusion.
The power to the device was turned off after 15 min.

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3.2. Cell exposure to continuous hyperthermia

The effects of continuous hyperthermia treatments were assessed
using clonogenic and trypan blue exclusion assays. First, a trypan blue
exclusion assay was used to analyze cellular growth under hyperthermic conditions. Briey, multiple samples of MDA-MB-231 breast cancer
cell line were cultured. Each sample set contained a minimum of four cell
culture asks. Once the sample sets had reached ~70% conuence, they
were 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 least
four 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 cell
viability/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 temperatures
of 37, 41, 43, or 45 C for up to 72 h. The cells were harvested immediately 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 plated into 6-well petri dishes, in order to plate 150 cells/well. They were
then allowed to grow into colonies for 710 days at 37 C, before xing
them with a 6% glutaraldehyde and 0.5% crystal violet mixture. The colonies were then counted with a colony counter pen. All the experiments
were performed in triplicates, and the results are reported as percentages of the colonies (% of control).
Insight into the effectiveness of continuous hyperthermia on cell
killing was given by the trypan blue exclusion assay, while cell reproductive 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 procedure was used to stimulate the potential effects of multiple heat shock therapy. Each cycle of treatment consisted
of 45 min of exposure to hyperthermic temperatures with in- and
between-cycle temperatures of 45 and 37 3 C, respectively. This
was followed by 12 h of incubation. During the heat exposure, a temperature gradient was achieved within the sample, with the center of
the dish at 4548 C and the edge of the dish at 3941 C, as discussed
above. The experiments were carried out for 5, 10 and 20 cycles. The
cell populations were then analyzed using both hemocytometry and
propidium iodine staining.
3.4. Propidium iodine assay for both cell death and apoptotic body
formation
Propidium iodine (PI) assays were used to determine the amount and
location of cell death within a sample. Cell death was assumed to be a
function of the local temperature, and thus a guide for the heat diffusion.
PI, at a concentration of 1 mg/mL in L15 medium supplemented with 10%
of FBS, was added directly to the samples, after a 12 h incubation period
following the last heat treatment. Subsequently, the samples were incubated for 15 min, allowing for the PI to stain and label the nuclei of the
dead cells. The cells were then xed with 3.7% formaldehyde for
15 min. Finally, the samples were observed and imaged using a Nikon
Eclipse 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 exposed
to heat shock in a circulating water bath for 15, 30, 45 and 60 min. The
samples were then rinsed in PBS, and xed in 3.7% formaldehyde solution in PBS for 15 min. After washing, the cells were stained and incubated with DRAQ5 (Biostatus Limited, Shepshed, Leicestershire, UK)
for nuclear staining for 5 min. The samples were then stained and

incubated with Oregon Green 488 paclitaxel (Invitrogen, Carlsbad, CA)

for tubulin labeling for 1 h. Subsequently, the cells were rinsed with
PBS and labeled for actin with FITC-conjugated phalloidin (Sigma, St.
Louis, MO) for 20 min. After several washes with PBS, the samples
were mounted on slides using a mounting medium, Aqua Poly/Mount
(Polysciences, Warrington, PA). A RS3 Spinning Disk Confocal microscope (Perkin Elmer, Waltham MA) with a 60 objective was then
used 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 shock
protein expression
To assess the effects of cyclic heating on heat shock protein expression, 2.5 million MDA-MB-231 cells were cultured into 6-well plates at a
concentration of 0.5 105 cells per mL. The cells were then grown for
24 h at 37 C and fed fresh medium prior to heat shock treatment. For
each heat shock treatment, cultured plate was heated at either 41 C,
43 C or 45 C for 30 min. This was accomplished by direct immersion
in a circulating water bath. The uniform bath temperatures serve as control experiments to simulate the effects of constant temperatures. The
temperatures in the medium were monitored with a PID controller. In
all cases, the culture plates reached the desired temperatures within
5 min. A 6-well plate was kept in the incubator at 37 C to represent
the control group. Medium was harvested from duplicate wells from
each plate immediately after treatment and 12 h after treatment. This
procedure was repeated every 12 h for 5 cycles to investigate the effects
of cyclic heating.
The number of cells was counted at each temperature to adjust the
results of the ELISA by the viable cell number. Two separate wells
were used exclusively for counting (to avoid collecting any viable cells
in the ELISA assay). These wells were harvested every 48 h and the
mean cell count and viability were determined by direct cell count in
a hemocytometer using the trypan blue exclusion assay.
To determine the concentration of inducible HSP70 in cells, an ELISA
assay was conducted following the manufacturer's instructions (R&D
Systems, Minneapolis, MN). Briey, a standard curve was created
using HSP70 standard solution. 100 L of standards and samples was
added to a 96-well plate and incubated at room temperature for 2 h.
Each well was washed with a wash buffer three times and then incubated with 100 L mAb against HSP70 at a concentration of 0.25 g/mL for
2 h at room temperature. Wells were again washed three times, and
100 L of Streptavidin-HRP (1:200 dilution from stock solution in buffered protein base) was added and incubated for 20 min at room temperature. Wells were washed three times and 100 L of substrate
solution (1:1 ratio of hydrogen peroxide to tetramethylbenzidine)
was added and incubated for 20 min at room temperature. Then, the reaction was stopped with 50 L of 2 N sulfuric acid, and the optical density 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 was
used to determine the statistical signicance between two samples
measuring the same variable. When comparing multiple treatments
with multiple factors, a multiple sample analysis of variance (ANOVA)
test was performed using Tukey's method. A p-value lower than 0.05
was considered a signicant difference, and condence intervals were
made 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

C. Theriault et al. / Materials Science and Engineering C 32 (2012) 22422249

to the cell culture medium. The model used Fourier's equation and a
nite difference scheme to predict the temperature gradient within
a plane of the implant. Briey, a 25 cm 2 square mesh was constructed
and divided uniformly into 10,000 nodes. An initial condition of 37 C
was imposed on the entire array at t = 0. Four boundary conditions
were considered: two in the x, two in the y-axis.
The boundaries were the contour of the implants and of the system. By assuming that the distance to the boundary of the system
was much larger than the size of the implant, heat diffusion in the furthest boundary region becomes negligible, allowing the coil boundary
temperatures to be set to 37 C. The steady-state temperature of each
node was then calculated using central difference and forward difference approximations of solutions to Fourier's laws.
4.2. Two-dimensional analysis of the heat diffusion from the hyperthermia
biocompatible device into a uid lled environment
To determine the temperature gradient in the area surrounding the
implant, the region was rst divided into an evenly distributed square
mesh. The grid consisted of 100 nodes dened along both x and yaxes, for an overall distribution of 10,000 total nodes. Each node was
represented by an area of x by y, as shown in Fig. 3.
The implant with square cross-sectional area of 1 cm 2 was considered to occupy 20 20 nodes in the center of the array. The nodes occupied by the implant were given initial conditions ranging from 45
to 55 C, while those outside of the system were set at 37 C. The
MATLAB program was then used to move forward in time, performing
iterations until steady-state temperature conditions were reached.
An energy balance was performed on each node using the nite
difference method (implementing the explicit method) in conjunction with the linear heat diffusion equation. The derivation for each
temperature equation is governed by a two-dimensional form of
Fourier's law of conduction. This is given by:
d2 T d2 T 1
1 dT

g
dt
dx2 dy2 k

2245

This expression can be rearranged to solve for the temperature at

node Ti,j at the time n + 1. This gives:

n1

T i;j t

T i;j

T ni1;j T ni1;j T ni;j1 T ni;j1 4T ni;j

!
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 direction
and two in the y direction) as well as 1 initial condition at t = 0. The
rst heat treatment for each sample was started after an incubation
period of about 12 h, and therefore, the initial temperature of the entire sample can be set at 37 C. Assuming a small temperature gradient between the implant and the initial temperature and large
implant-to-boundary distance, all nodes on the outer edges of the
grid 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 experiments
are presented in Fig. 4(a) and (b). Both gures show cell culture
data that was obtained from MDA-MB-231 breast cancer cell incubated for up to 72 h at constant hypothermic conditions. Fig. 4(a) shows
the growth rate of this particular cell line at 37 C, as well as the severe decrease in growth, as the temperature increased. Necrosis is observed above 43 C, which is similarly reported for other cell lines
[4952]. Treatment at 43 C is shown to be the optimal temperature
for hyperthermia treatments, which is consistent with reports in the
literature [53]. Fig. 4(b) shows similar trends for colony growth.

a
37C

T ni1;j 2T ni;j T ni1;j

x2

T ni;j1 2T ni;j T ni;j1

y2

n1
n
1 T i;j T i;j
t

Normalized Cell Growth

100%

where g is dened as the heat generation term, and k and are the
thermal conductivity and thermal diffusivity of the medium, respectively. Neglecting the heat generation term and applying a forwardtime, central difference discretization for an interior node with no
heat generation yields:

50%

41C
0%
0

12

24

36

48

60

72

-50%
43C
45C

-100%

Time (hr)

Number of Colonies (% of Control)

41C

100

43C

75

50

25

45C

24

48

72

Time (Hours)

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.

Fig. 4. Effect of different temperatures of continuous hyperthermia on MDA-MB-231

Cell Population. Breast cancer cells MDA-MB-231 were incubated continuously at either 37, 41, 43, or 45 C for 72 h or until the population collapsed. Results are shown
using a) trypan blue exclusion assay and b) clonogenic assay.

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C. Theriault et al. / Materials Science and Engineering C 32 (2012) 22422249

5.2. Comparison of measured and predicted temperatures proles of

device mediated hyperthermia
In-vitro experimental observation showed that steady-state temperature conditions were achieved in a very short period of time
(less than 4 min). It was, therefore, determined that the transient response was not very signicant. Consequently, only the steady-state
temperature distributions were examined. Typical results from the
temperature modeling are presented in Fig. 5(a). These show that
the temperatures decrease with increasing distance from the center
of the device. The modeling results were supported by the in-vitro experiments. These were found to be in close agreement with the
predicted temperatures from the MATLAB simulation, as shown in
Fig. 5(b).

5.3. Staining of cell death and cell morphology under isothermal

hyperthermia
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) to
evaluate the effects of the heat treatment on the cell populations.
Using our implantable device, the center of the sample was brought
to 55 C, while the outside edge remained below 41 C. Visual inspection of PI staining of these samples showed the presence of both necrotic and apoptotic cell death, with necrosis prevalent in the region

of high-hyperthermia (>45 C) and the apoptotic cell death in the

lower-temperature regime (Fig. 6). This result is consistent with previous studies linking necrotic cell death with temperatures in the
upper-hyperthermic range and apoptotic cell death with temperatures in the lower-hyperthermic range [11,52,54,55].
Confocal microscopy images of cell samples exposed to 15, 30, 45
and 60 min of hyperthermia at 43 C are presented in Fig. 7. The samples were xed and stained for cytoskeletal structures including actin,
microtubules, and nucleus. No signicant difference was observed between the cytoskeletons of the control and the samples exposed for
15 min at 43 C. The most signicant changes were observed in the
microtubule network. Specically, after 30 min of exposure, changes
in the microtubule networks were observed. Some condensation of
the 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 observed
at 45 and 60 min. Actin networks were more disorganized after
60 min, compared to those that were observed after 45 min.
Nuclear changes were present in approximately one third of the
samples exposed for 60 min. For those that did exhibit some nuclear
changes, mitotic body formation could be observed; presumably indicating that these cells had entered apoptosis. These results clearly
show that after 30 min of exposure to 43 C, the cells started to experience signicant deterioration of their cytoskeleton, which continued with increased heat exposure.
5.4. Cyclic hyperthermia

a 100

54

90

52

80

50
70

48
60

46

50
40

44

30

42

20

40

10

38
10

20

30

40

50

60

70

80

90

100

Fig. 5. Temperature variation radiating from hyperthermia implant. Results are shown
from (a) modeling the thermal diffusion from the hyperthermia device into its surroundings using MATLAB. The implant is set at 55 C and the outside extremities
were set to 37 C (scale: 10 unit = 0.5 cm). The results from the model (blue) are compared with the average experimental measurements (red) in (b).

The results of the cyclic hyperthermia experiments are presented in

Fig. 8(a) and (b). There is a statistically signicant difference between
the control and hyperthermia treatment at all cycles (pb 5 10 5). Although there is a decline in cell population after 5 hyperthermia treatment cycles, statistical analysis revealed that it was not signicant. In
fact, the analysis showed that there was no signicant difference between the mean number of cells at the beginning of the experiments
and after 5 or 10 cycles. However, after 20 cycles, a statistically signicant decrease was observed in the cell populations, when compared
to the other treatment cycles.
5.5. In-vitro cyclic heating and heat shock protein expression
The PI staining showed clearly that cell death propagated radially
outwards from the device after cyclic hyperthermia (Fig. 8(b)). Similar trends were observed in the cell death proles after isothermal
heating at 43 C (Fig. 6). The most prominent form of cell death
seems to be apoptosis, as demonstrated by the numerous apoptoticbodies present in the samples. These results also show that at higher
temperatures, fewer cycles are needed to induce cell death. Moreover, the results also suggest that cell-cycle arrest was achieved within a large range of temperatures, ranging from 41 to 47 C. The results
show that heat shock protein expression increases with increasing
temperature between 41 and 47 C (Fig. 9).
Prior work has shown that heat shock proteins are proteins that are
over-expressed during hyperthermia. In most tumor cells, they are already over-expressed when compared to the basal level in normal cells
[52,5658]. Recent work suggests that HSPs have the ability to serve as
carriers of tumor antigens and inammatory agents. Specically, multiple studies have shown that HSP70 interacts with receptors on antigen
presenting cells and can mediate T-lymphocytes to trigger an immune
response against the cells that are expressing HSP70 [57,59,60]. A
major determinant of immune response is the interaction between the
antigen-presenting cells and the T lymphocytes [61] due to its vasodilatation properties. The current results (Fig. 9) show that the extent of HSP
expression increases with increasing temperature. The above interactions are, therefore, likely to increase with increasing temperature, for
temperatures between 41 and 47 C.

C. Theriault et al. / Materials Science and Engineering C 32 (2012) 22422249

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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 uorescent labeling was assayed using 488 nm excitation light source. Shown above are images from three regions of interest: the center (A), the position (B), and the edge (C). Scale bars in
bottom 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 as
viewed under regular light microscopy. All three images show a similar cell distribution pattern, indicating a homogeneous cell distribution. A2, B2, and C2 show the PI stain indicating the
amount 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 marker
for cellular death, towards the middle of the sample.

6. Discussion
6.1. Morphological changes in the cell cytoskeleton as a potential cause
for both the disturbance of cellular function and for the cell's thermosensitive properties
Prior research has shown that heat shocks can have negative effects on the cell cycle, often causing a temporary arrest that can last
from 2 to 14 h [52,62]. Our results suggest that, after 5 days of cell

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 mitotic block. Although more work is needed, our immuno-urescence results do support the hypothesis of mitotic arrest as the primary
trigger 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 the
microtubules structures, or in another regulatory protein, hence interfering with normal polymerization mechanisms and blocking the

Fig. 7. Cytoskeleton of MDA-MB-231 cells exposed to hyperthermia for different amount of time. Confocal microscopy was performed on samples exposed to 43 C for 0, 15, 30, 45,
and 60 min. No signicant differences could be observed in the samples that were exposed to 43 C for 15 min compared to the control. After 30 min of hyperthermia exposure,
signicant aggregation of the microtubule network could be observed which increased with continued exposure. Differences in the actin laments could be observed starting at
30 min of exposure. At 60 min no clear actin network organization could be observed in the majority of samples. Nuclear deterioration in the form of mitotic bodies (as shown
above) was only observed in about one third of cells after 60 min of exposure.

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C. Theriault et al. / Materials Science and Engineering C 32 (2012) 22422249

6.2. The biocompatible hyperthermia device and its potential for medical
applications

Fig. 8. Growth as a function of heat shock cycles. (a) MDA-MB-231 cell samples were
exposed to multiple cycles of a heat shock duration of 45 min followed by a relaxation
period of 12 h. Cell growth arrest was observed for the rst 10 cycles with a small nonstatistically signicant decrease in the cell population after 10 cycles. After 20 cycles of
heat shock treatment, a large statistically signicant drop (p b 5 10 5) in the cell population was observed. The underlying cause of cell death was hypothesized to be the
prolonged cell cycle arrest caused by the cyclic heat shock as 45 min of exposure to
43 C was shown to be non-lethal on its own. (b) PI staining of MDA-MB-231 cells
showed a radial pattern of cell death: (bA) shows the cell population and (bB) shows
the cells that have died in the population. The hyperthermia device is located at the extreme right of the gure. This result was similar to the pattern observed during continuous hyperthermia exposure. Scale bar in lower right corresponds to 500 m.

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

autonomy

200

In order for a hyperthermia device to be clinically efcient, there is

a need for it to achieve a certain level of power autonomy. One potential design would be to create an implant consisting of a closed coil
embedded in PDMS. By introducing an alternating magnetic eld
(AMF) in its vicinity, a current could be induced, thereby enabling resistive heating. The surrounding temperature would also have to be
monitored and controlled using wireless technology to maintain hyperthermic temperatures. One potential pitfall with this system includes the fact that the implant coil would have to be oriented in a
specic direction within the body for greatest power efciency.
In any case, the resulting current ow could then be stored in a rechargeable battery to provide an autonomous power source. Further
work is clearly needed to demonstrate the feasibility of such systems.
These should be combined with efforts to develop autonomous power
supplies for sustained use in the human body. More advanced temperature controls are also warranted. Finally, future versions of the
device should be capable of automatically delivering the right amount
of heat at the correct intervals without human interaction.

150

6.4. Potential future directions

cell at the mitotic spindle assembly checkpoint. Recently, Michalakis

et al. [63] showed very similar results pertaining to the microtubule
organization of HeLa cells after 1 h exposure to a 39 C heat shock.
Their research also showed similarity between cells treated with
heat shock and tubulin inhibitor (paclitaxel, arsenide, etc), indicating
that mitotic arrest is the most likely the major apoptosis pathway.

250
Heat Shock Protein (ng per 106cells)

Although it is clear that this device could be used for treating a wide
range of cancers, this work suggests that initial attention could be paid to
breast cancer, specically in patients with stage II or III non-metastatic
tumors. In such scenarios, surgical removal of the tumor is normally possible, and radiotherapy as well as low doses of chemotherapy are still
prescribed in an effort to quench the body of any remaining cancer
cells and improve the patient's prognosis. As an alternative, the hyperthermia device developed in this study could be implanted during the
surgical removal of a tumor. The device could then not only serve as a
stand-alone treatment modality, but could be used in combination
with either radiotherapy or chemotherapy. Moreover, multiple studies
[34,64] have shown additive and even synergistic results when hyperthermia is combined with radiotherapy.
Prior work on internal heat and pain receptors have also shown that
sub-dermal temperatures of up to 43 C are very well tolerated clinically, with patients feeling only a slight discomfort [65]. Furthermore, the
blood perfusion rate, convection due to surrounding arteries, and uniform thermal values could be taken into account using the bio-heat
transfer equations [66,67]. Under such conditions, the heat ow can
be described by a three-dimensional (3D) heat diffusion model rather
than the two-dimensional version presented here. A 3D model could,
therefore, be used to predict which regions of the surrounding tissue
will reach hyperthermic temperatures by taking into account the actual
patient-specic data retrieved from clinical imaging techniques, such as
MRI and ultrasound.

37C

100

41C
43C
45C

50

0
T0

T12
HS1

T0

T12
HS2

T0

T12
HS3

T0

T12
HS4

Heat Shock Trial (12hr Intervals)

Fig. 9. Heat shock protein assay results. The extent of HSP expression from MDA-MB-231
cells increases with increasing temperature.

Future work is needed to determine the response of normal breast

cells, as well as other cancer cell lines, to the effects of heat. A recent
study by Rylander et al. [68] showed that the HSP expression prole of
cancerous prostate cells was more sensitive to hyperthermia than their
non-cancerous counterparts. In future work, uorescence-activated cell
sorting (FACS), using propidium iodine or Annexin V, could be used to
explore the portions of cells that are in early or late stage apoptosis, or
necrosis. Furthermore, FACS cell cycle analyses should reveal at what
point hyperthermia causes mitotic arrest. Additionally, further quantication of the cellular HSP expression prole in relation to cellular proximity to the hyperthermia-inducing device could provide additional
insights.

C. Theriault et al. / Materials Science and Engineering C 32 (2012) 22422249

Furthermore, since the cellular responses of 2D models have been

shown to be different from 3D in-vivo models, there is a need to explore the response to heat in more complex 3D in-vitro or in-vivo
models that simulate tumor conditions. This could be done in 3D
micro-environments or in-vivo models that could further investigate
the use of regular short hyperthermia pulses in the selective killing
of cancer cells. Such experiments could be performed with our without the proposed hyperthermia delivery device.
Also, although the current results do indicate that, in a simple 2D
cell culture environment, a pulsed hyperthermia regimen was efcacious in halting cellular growth in MDA-MB-231 cells, in-vitro 3D
micro-environments can be simulated using gels and agars to mimic
tissue-like structures. The relationships between cell viability and distance from the device should also be modeled quantitatively [69]
after heat exposures in such 3D micro-environments. These are clearly some of the challenges for future work.
7. Summary and concluding remarks
This paper describes the results of an experimental and computational study of the effects of localized hyperthermia on breast cancer
cells. Controlled hyperthermia was achieved using an implantable device. The results from the in-vitro studies show clearly that low levels
of hyperthermia, when delivered in a short pulse regimen, can cause
cell cycle arrest in tumor cells. This leads to the inhibition of cell growth
and increasing cell death after more than ve days of cyclic hyperthermia. The observed increase in cell death is also associated with heat
shock protein expression. This study suggests that, after 5 days of cyclic
heat treatment, using the implantable device, MDA-MB-231 cells undergo cell cycle arrest.
The current results clearly suggest a need for in-vivo testing. Furthermore, there is a need for additional studies that use surface texture to
promote the improved integration of the device with biological tissue.
Acknowledgments
This work was supported by the National Science Foundation (Grant
No. DMR 0231418), the Grand Challenges Program at Princeton University and the sponsor-id="gs3" id="gts0015">STEP-B Program of the
World Bank. The authors are grateful to these organizations for their
support of the research.
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