होम Radiology Orthotopic Esophageal Cancers: Intraesophageal Hyperthermia-enhanced Direct Chemotherapy in Rats

Orthotopic Esophageal Cancers: Intraesophageal Hyperthermia-enhanced Direct Chemotherapy in Rats

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भाषा:
english
पत्रिका:
Radiology
DOI:
10.1148/radiol.2016152281
Date:
July, 2016
फ़ाइल:
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Original Research

Yaoping Shi, MD, PhD2
Feng Zhang, MD, PhD
Zhibin Bai, MD
Jianfeng Wang, MD, PhD
Longhua Qiu, MD, PhD
Yonggang Li, MD, PhD
Yanfeng Meng, MD, PhD
Karim Valji, MD, PhD
Xiaoming Yang, MD, PhD

Purpose:

To determine the feasibility of using intraesophageal radiofrequency (RF) hyperthermia to enhance local chemotherapy in a rat model with orthotopic esophageal squamous cancers.

Materials and
Methods:

The animal protocol was approved by the institutional animal care and use committee and the institutional review
board. Human esophageal squamous cancer cells were
transduced with luciferase lentiviral particles. Cancer
cells, mice with subcutaneous cancer esophageal xenografts, and nude rats with orthotopic esophageal cancers
in four study groups of six animals per group were treated
with (a) combination therapy of magnetic resonance imaging heating guidewire–mediated RF hyperthermia (42°C)
plus local chemotherapy (cisplatin and 5-fluorouracil), (b)
chemotherapy alone, (c) RF hyperthermia alone, and (d)
phosphate-buffered saline. Bioluminescent optical imaging
and transcutaneous ultrasonographic imaging were used
to observe bioluminescence signal and changes in tumor
size among the groups over 2 weeks, which were correlated with subsequent histologic results. The nonparametric Mann-Whitney U test was used for comparisons of
variables.

Results:

Compared with chemotherapy alone, RF hyperthermia
alone, and phosphate-buffered saline, combination therapy with RF hyperthermia and chemotherapy induced the
lowest cell proliferation (relative absorbance of formazan:
23.4% 6 7, 44.6% 6 7.5, 95.8% 6 2, 100%, respectively;
P , .0001), rendered the smallest relative tumor volume
(0.65 mm3 6 0.15, P , .0001) and relative bioluminescence optical imaging photon signal (0.57 3 107 photons
per second per square millimeter 6 0.15, P , .001) of
mice with esophageal cancer xenografts, as well as the
smallest relative tumor volu; me (0.68 mm3 6 0.13, P ,
.05) and relative photon signal (0.56 3 107 photons per
second per square millimeter 6 0.11. P , .001) of rat
orthotopic esophageal cancers.

Conclusion:

Intraesophageal RF hyperthermia can enhance the effect
of chemotherapy on esophageal squamous cell cancers.

1

From the Image-guided Biomolecular Intervention
Research and Section of Vascular and Interventional Radiology, Department of Radiology, University of Washington
School of Medicine, Campus Box 358056, 850 Republican
St, Room S470, Seattle, WA 98109. Received October 16,
2015; revision requested December 14; revision received
March 29, 2016; accepted April 18; final version accepted
April 26. Address correspondence to X.Y. (e-mail:
xmyang@u.washington.edu).
F.Z. and X.Y. supported by Society of Interventional
Radiology (Pilot Research Grant). F.Z. and X.Y. supported by
National Institutes of Health (RO1EBO12467).

q

Current address:
2
Department of Tumor Interventional Treatment, Renji
Hospital, School of Medicine, Shanghai Jiaotong University,
Shanghai, China.

n Experimental Studies

Orthotopic Esophageal Cancers:
Intraesophageal Hyperthermiaenhanced Direct Chemotherapy in
Rats1

RSNA, 2016

Y.S. and F.Z. contributed equally to this work.
q

RSNA, 2016

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EXPERIMENTAL STUDIES: Hyperthermia-enhanced Direct Esophageal Tumor Chemotherapy

E

sophageal cancer remains one of
the leading causes of cancer-related mortality, with an estimated
455 800 new cases and 400 200 deaths
per year worldwide (1,2). Surgery offers limited benefits for patients with
advanced esophageal cancers, with
extremely poor overall 5-year survival
ranging from 15% to 25% (1,3,4). Radiation therapy as a palliative treatment is even less effective than surgical resection and is associated with a
high likelihood of major complications
such as tracheoesophageal fistula (1).
Recent efforts have been focused on
combination treatment with surgery,
radiation therapy, and systemic chemotherapy to achieve better local
control of disease and improve overall
survival of patients with this deadly
disease (1). However, randomized
clinical trials have shown mixed results with respect to progression-free
or overall survival benefit with such
combination therapy (1,4).
It is well known that systemic chemotherapy is often unable to deliver
sufficient chemotherapeutic drugs into
target tumors and is often plagued by
low chemotherapeutic sensitivity, resistance to chemotherapy, and high
risk of systemic toxicity to other vital organs (5–7). On the other hand,
imaging-guided minimally invasive
interventional oncologic techniques

Advance in Knowledge
nn Combination therapy with MR
imaging and heating guidewire–
mediated radiofrequency (RF)
hyperthermia and chemotherapy
induced the lowest cell proliferation, rendered the smallest relative tumor volume and relative
bioluminescence optical imaging
photon signal in mice with
esophageal cancer xenografts,
and resulted in the smallest relative tumor volume and relative
photon signal in rat orthotopic
esophageal cancers compared
with groups treated with chemotherapy alone, RF hyperthermia
alone, and phosphate-buffered
saline.
2

Shi et al

enable delivery of high doses of chemotherapeutic agents into target tissue
with improved tumoricidal activity but
fewer systemic adverse effects (8,9).
Recent studies have confirmed that
nonablative hyperthermia at approximately 42°C can significantly enhance
the sensitivity of cancer cells to chemotherapeutic drugs and reverse chemotherapy resistance (10–12). In current
clinical trials, nonablative hyperthermia for treatment of malignancies is
generated by means of either systemic
whole-body or external hyperthermia
(13,14). However, because of the deep
anatomic location of the esophagus,
which is surrounded by vital organs including the heart, lungs, trachea, and
spine, it is difficult to generate highly
focused heat only at the site of esophageal tumors by using systemic or external hyperthermia. Generation of
external hyperthermia in the esophagus poses the risk of injury to adjacent
vital organs, such as the spinal cord
(15,16). In the past decade, we have
developed a U.S. Food and Drug Administration–approved magnetic resonance (MR) imaging heating guidewire
that can be used not only for highspatial-resolution luminal wall imaging
and guiding interventions but also may
be applied as an intraluminal heating
source for enhancing local gene and
chemotherapy treatment (8,17,18).
The purpose of this study was to determine the feasibility of using intraesophageal radiofrequency (RF) hyperthermia to enhance the effect of local
chemotherapy in rats with orthotopic
esophageal squamous cancers.

Materials and Methods
Study Design
We divided the study into three stages:
(a) in vitro evaluation with the use of
human esophageal squamous cancer
cells to establish “proof-of-principle”
of the new concept of RF hyperthermia–enhanced chemotherapy, (b) in
vivo confirmation of this new concept
in a mouse model with subcutaneous
esophageal squamous cancer xenografts, and (c) preclinical validation of
the feasibility of the new technique in
a rat model of molecular imaging–detectable orthotopic esophageal cancer.

In Vitro Evaluation
Cell culture and RF hyperthermia–
enhanced killing effect of chemotherapeutic drugs.—Human esophageal
squamous cancer cells (JCRB Cell Bank,
Osaka, Japan) were transduced with luciferase–red fluorescence protein–lentivirus gene to create luciferase- and red
fluorescence protein–positive esophageal squamous cancer cells according to
the manufacturer’s protocol (GeneCopoeia, Rockville, Md). Luciferase- and
red fluorescence protein–positive cells
were sorted by using a fluorescenceactivated cell sorting technique (Aria II;
Becton Dickinson, Franklin Lakes, NJ).
Cells were then seeded in four-chamber

Published online before print
10.1148/radiol.2016152281 Content codes:
Radiology 2016; 000:1–10
Abbreviations:
PBS = phosphate-buffered saline
RF = radiofrequency

Implication for Patient Care
nn RF hyperthermia can enhance
the effect of local chemotherapy
with cisplatin and fluorouracil
in rat orthotopic esophageal
cancers, which indicates that
intraesophageal RF hyperthermia–enhanced local chemotherapy shows potential for development as an alternative
treatment for esophageal
cancers.

Author contributions:
Guarantors of integrity of entire study, Y.S., F.Z., Z.B., X.Y.;
study concepts/study design or data acquisition or data
analysis/interpretation, all authors; manuscript drafting or
manuscript revision for important intellectual content, all
authors; approval of final version of submitted manuscript,
all authors; agrees to ensure any questions related to the
work are appropriately resolved, all authors; literature
research, Z.B., Y.M.; experimental studies, Y.S., F.Z., Z.B.,
J.W., L.Q., Y.L., Y.M.; statistical analysis, Y.S.; and manuscript editing, Y.S., K.V., X.Y.
Conflicts of interest are listed at the end of this article.

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cell culture slides (NalgeNunc International, Rochester, NY) and maintained
in Delbecco’s modified Eagle’s medium
and F12 (1:1), supplemented with 10%
fetal bovine serum (Gibco, Grand Island, NY). RF hyperthermia was performed as described in the literature
(18). RF hyperthermia was performed
by placing a 0.022-inch MR imaging
heating guidewire under the bottom of
chamber four of the chamber slides.
A 400-mm fiber optical temperature
probe (PhotonControl, Burnaby, British Columbia, Canada) was placed in
the chamber for temperature measurement. By adjusting RF output power at
approximately 10 W, the temperature
was kept at 41 °C 6 1. Cells in different
groups were treated with (a) cisplatin
(4.5mg/mL) and fluorouracil (15mg/mL)
plus 30 minutes of RF hyperthermia at
41 °C 6 1; (b) cisplatin (4.5mg/mL) plus
fluorouracil (15mg/mL) alone; (c) 30
minutes of RF hyperthermia alone; and
(d) phosphate-buffered saline (PBS) to
serve as a control group. We used the
30% inhibitory concentration doses of
cisplatin and fluorouracil for cell treatment, which were determined by means
of MTS assay (3-[4,5-dimethylthiazol2-yl]-5-[3-carboxymethoxyphenyl]-2-[4sulfophenyl]-2H-tetrazolium, CellTiter
96 Aqueous One Solution Cell Proliferation Assay; Promega, Madison, Wis).
Cisplatin and fluorouracil are the firstline chemotherapeutic drugs for treating patients with esophageal cancers
(19).
Cell proliferation assay.—Cell proliferation was evaluated by means of
MTS assay 48 hours after the treatments. Relative cell proliferation of
different cell groups was calculated by
using the equation Atreated 2 Ablank/Acon2 Ablank, where A is absorbance of
trol
formazan. Formazan is the bioreduced
product of MTS by viable cells. Cells on
cell culture slides were subsequently
washed twice with PBS, fixed in 4%
paraformaldehyde, counterstained with
4’,6-diamidino-2-phenylindole (DAPI;
Vector Laboratories, Burlingame, Calif), and then imaged with a fluorescence microscope. All experiments for
each of the cell groups were repeated
six times.
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Apoptosis assay.—The percentages
of viable and apoptotic cells were determined by means of flow cytometry
with Annexin V-fluorescein isothiocyanate and propidium iodide (BD
Biosciences, San Diego, Calif). Cells
were stained with the staining kit in
a binding buffered solution along with
the appropriate control solution. The
total number of fluorescein and propidium–positive cells were counted by
using a flow cytometer (FACScan; BD
Biosciences). The data were analyzed
by using software (FlowJo version 10;
FloJo Data Analysis Software, Ashland, Ore).

In Vivo Confirmation
Creation of animal models.—The animal protocol was approved by our
institutional animal care and use
committee. The animals were anesthetized with 1%–3% isoflurane (Piramal
Health care, Andhra Pradesh, India) in
100% oxygen. For creation of mouse
models with subcutaneous esophageal
squamous cancer xenografts, we used
24 nude mice aged 4–6 weeks (NU/
NU mice; Charles River Laboratories,
Wilmington, Mass). Each mouse was
inoculated subcutaneously in the unilateral back with luciferase- and red fluorescence protein–positive embryonic
stem cells (5 3 106 to 1 3 107) in 100
µL of an extracellular matrix (Matrigel;
Corning Life Sciences, Corning, NY).
Once the size of the tumor reached
5–10 mm in diameter, we began the experimental procedures.
For creation of rat models with
orthotopic esophageal squamous cancers, we used 24 nude rats (weight,
180–220 g), and all procedures were
performed by using a real-time ultrasonograpic (US) imaging–guided minimally invasive approach. The nude rats
were positioned supine on the surgical table. A 0.035-inch guidewire was
transorally introduced into the esophagus, and then a custom microcatheter
was advanced into the cervical esophagus over the guidewire. After the
guidewire was withdrawn, a custom
microcoaxial needle that had a curved
tip was positioned into the target
esophagus through the microcatheter.

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Under real-time US imaging guidance,
the curved needle tip was advanced
into the cervical esophageal wall with
a controlled penetration depth of 3
mm, at which point, 5 3 106 to 1 3
107 luciferase- and red fluorescence
protein–positive esophageal squamous
cancer cells in 100 µL of the gelatinous
protein mixture were injected into the
target esophageal site.
In vivo confirmation of the concept:
RF hyperthermia–enhanced chemotherapy in a mouse model with subcutaneous esophageal squamous cancer
xenografts.—The 24 mice with esophageal squamous cancer xenografts were
randomly allocated into four groups.
Six mice in each of the four groups
were treated with (a) direct intratumoral injections of 2 mg/kg of cisplatin
and 25 mg/kg of fluorouracil in 100 µL
of PBS, immediately followed by local
RF heating at 41 °C 6 1 for 30 minutes;
(b) intratumoral injection of 2 mg/kg of
cisplatin and 25 mg/kg of fluorouracil
in 100 µL of PBS alone; (c) 30 minutes
of RF hyperthermia alone; or (d) intratumoral injection of 100 µL of PBS
to serve as the controls. A Hamilton
microsyringe was used for injection of
the chemotherapeutic drugs at a rate of
100 µL per 3 minutes. RF hyperthermia
was performed as described in the literature (8). RF heating was performed
by inserting a 0.022-inch MR imaging
heating guidewire into the tumor, with
its hyperthermia source at the center
of the tumor. A 400-mm fiber optical
temperature probe was placed subcutaneously parallel to the MR imaging
heating guidewire at the margin of the
tumor to measure the temperature.
In vivo validation of the new technique: intraesophageal RF hyperthermia–enhanced direct intratumoral
chemotherapy in a rat model with
orthotropic esophageal squamous cancers.—When the volume of tumors
grew to approximately 50 mm3, chemotherapeutic drugs were directly
injected into the esophageal tumor
through the intraesophageal delivery
needle under US imaging guidance at
a rate of 100 uL per 3 minutes. We
used the same concentration of cisplatin and fluorouracil as was used in
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EXPERIMENTAL STUDIES: Hyperthermia-enhanced Direct Esophageal Tumor Chemotherapy

mice, and chemotherapeutic drug solutions at 100 mL per 100 mm3 tumor
were injected into tumors. A Hamilton
microsyringe was used for injection of
the chemotherapeutic drugs at a rate
of 100 mL per 3 minutes. Immediately
after drug delivery, intraesophageal RF
hyperthermia was generated by inserting a 0.022-inch MR imaging heating
guidewire into the esophagus with its
heating element centered in the tumor.
The MR imaging heating guidewire was
made of a coaxial copper cable with a
3-cm extension of its inner conductor.
When external RF thermal energy is
delivered, the wire can create a very
localized and controlled heat source,
with the heating core at the junction
of the inner conductor and outer conductor of the wire (19). The RF power
distribution is homogeneous within a
distance of 1 cm around the heating
spot of the wire (20,21). A fiber optical temperature probe was placed in
the esophagus next to the MR imaging heating guidewire to allow simultaneous monitoring of temperature. The
intraesophageal RF heat was set at 41
°C 6 1 for 30 minutes.
Posttreatment follow-up with optical and US imaging.—We used optical
imaging to follow up on tumor response
at days 0, 7, and 14 after treatment.
Optical imaging was conducted with
an MR imaging system (Bruker In
vivo Xtreme; Bruker, Billerica, Mass).
Each animal was imaged at day 0 before treatment and days 7 and 14 after
treatment. For mice, optical imaging of
red fluorescence protein–positive tumors was acquired with an excitation
wavelength of 570 nm and emission
wavelength of 600 nm. For rats, optical
images were achieved 20 minutes after
an intraperitoneal injection of d-luciferin at 150 mg/kg (Pierce D-Luciferin;
ThermoFisher Scientific, Rockford, Ill).
Signal intensity was quantified by using
the Bruker software. Relative signal intensity (RSI) was calculated by using
the following equation: RSI = SIDn/SID0,
where SI is signal intensity, Dn represents days after treatment, and D0 is
the day before treatment.
US imaging was then performed to
assess tumor growth (Sonosite; Bothel,
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Shi et al

Wash) at days 0, 7, and 14 after treatment. The axial (x) and longitudinal
(y) diameters of tumors and tumor
depths (z) were measured on the US
images with maximal tumor sizes. The
volume of each tumor was then calculated according to the following equation for volume: v = x · y · z · p/6.
Data were expressed as relative tumor
volume (RTV) by using the following
equation: RTV = VDn/VD0, where V is
tumor volume, Dn represents days after treatments, and D0 is the day before treatments.
Histologic correlation and confirmation.—Since this study was focused primarily on the new technical
development, we did not continue
following up until complete tumor disappearance. Tumors were harvested
at day 14 after treatment. Tumor tissue was embedded in optimal cutting
temperature compound, frozen in liquid nitrogen, kept frozen at 280°C,
and then cryosectioned into 10-µm
slices, which were placed on slides
for apoptosis staining. The level of
apoptosis was determined by means
of terminal deoxynucleotidyltransferased deoxyuridine triphosphate nickend labeling assay by using a kit (TACS
XL Blue Label kit; Trivegen, Gaithersburg, Md) according to the manufacturer’s instructions. Cells with dark
blue dots in the cytoplasm were recognized as apoptotic cells. On each slide,
six high-powered fields were pictured
randomly by using a digital camera
(DP72; Olympus, Tokyo, Japan). Results were expressed as the apoptotic
index (AI), which was calculated by
using the following equation: AI = AC/
TC · 100%, where AC is the number
of apoptotic cells and TC is the total
number of cells.

Statistical Analysis
Statistical software (SPSS 19.0; SPSS,
Chicago, Ill) was used for all data
analyses (8). A nonparametric MannWhitney U test was used to compare
(a) relative proliferation rates among
different cell groups; (b) relative optical
signal intensity values; and (c) relative
tumor volumes at different time points
among the animal groups with various

treatments. A P value of less than .05
was considered to indicate a significant
difference.

Results
In Vitro Evaluation: RF Hyperthermia–
enhanced Chemotherapeutic Effect on
Esophageal Squamous Cancer Cells
Confocal microscopy performed 48
hours after treatment demonstrated
diminished cell survival with combination therapy compared with that in the
other three treatment groups (Fig 1a).
Quantitative MTS assay allowed further confirmation that cell proliferation with combination therapy (relative absorbance of formazan: 23.4%
6 7) was significantly lower than that
seen in the groups treated with chemotherapy alone, RF hyperthermia alone,
and PBS (44.6% 6 7.5, 95.8% 6 2,
and 100%, respectively; P , .0001)
(Fig 1b). After staining the cells with
Annexin V-FITC/PI, we used flow cytometry to quantify apoptosis in the
four study groups. Flow cytometry results confirmed the results of confocal
microscopy and MTS assay, showing
more apoptotic cells in the combination therapy group than in the other
study groups (Fig 1c).
In Vivo Confirmation: RF Hyperthermia–
enhanced Chemotherapy in a Mouse
Model with Esophageal Squamous Cancer
Xenografts
All animals survived the experimental
procedures without complications. Follow-up US imaging demonstrated the
smallest relative tumor volume in the
combination therapy group (0.65 mm3
6 0.15) compared with those in the
other three groups (1.28 mm3 6 0.12,
2.67 mm3 6 0.48, and 2.71 mm3 6
0.3, respectively; P , .0001) (Fig 2a,
2c). Optical imaging was used to follow
tumor response to the treatments, and
it demonstrated significantly lower relative photon signal in the combination
therapy group (0.57 3 107 photons per
second per square millimeter 6 0.15)
compared with that in the chemotherapy-only group (1.38 3 107 photons
per second per square millimeter 6

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Figure 1

Figure 1: (a) Confocal photomicrograph shows the lowest cell survival in
the group receiving combination therapy (chemotherapy and RF hyperthermia),
compared with those in the other three treatment groups. (b) MTS assay
further shows the lowest cell viability (∗ = P , .0001). (c) Apoptosis analysis
confirms more apoptotic cells in the combination therapy group than in the
other three groups. Comp-PE-A = compensation-phycoerythrin-adjustment,
Comp-FITC-A = compensation-fluorescein-isothiocyanate-adjustment, RFH =
radiofrequency heat.

0.3; P = .0003), RF hyperthermia–
only group (2.4 3 107 photons per
second per square millimeter 6 0.47,
P , .0001), and PBS group (2.56 3
107 photons per second per square
millimeter 6 0.63, P , .0001) (Fig
2b, 2d). Histologic analysis of tumors
showed the smallest tumor (Fig 3,
upper panel) and a higher apoptotic
index in the group treated with combination therapy (46.5% 6 13.6) than
in the groups treated with RF hyperthermia only (7.8% 6 2.2, P , .001)
or chemotherapy only (24.2% 6 8.1, P
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= .0063) (Fig 3, bottom panel), which
was correlated with subsequent imaging findings.

Intraesophageal RF Hyperthermiaenhanced Direct Intratumoral
Chemotherapy of Orthotopic Esophageal
Squamous Cancers
We designed and manufactured a microintraesophageal agent delivery and RF
hyperthermia heating system (Fig 4, A),
which enabled us to (a) locally implant
esophageal squamous cancer cells into
the target esophageal segment, (b)

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deliver chemotherapeutic agents directly into the developing esophageal
tumors, and (c) transfer RF heat within
the esophageal lumen to locally enhance
chemotherapy of the target tumors.
Under real-time US imaging guidance,
we inoculated the wall of the cervical
esophagus with a luciferase- and red
fluorescence protein–positive esophageal squamous cancer cell suspension
by means of an intraesophageal approach. US imaging allowed detection
of the cell pellet in the tissue adjacent
to the wall of the esophagus (Fig 4, B).
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Figure 2

Figure 2: (a) US images and (b) optical and x-ray images show tumor growth and responses to treatments at days 0, 7, and 14. There was a statistically significant
decrease in both relative tumor volume (arrows on a; graph c) and fluorescent signal intensity (yellow-red color on b, graph d) with combination therapy (chemotherapy and RF hyperthermia) compared with the other three treatment groups. RFH = radiofrequency heat.

Approximately 3 weeks later, both US
and optical imaging demonstrated the
presence of a tumor mass in the tissue
(Fig 4, C, D).
All animals survived all experiments without obvious complications.
6

Examinations of gross specimens obtained at the end of the experiments
revealed tumor adherent to the wall
of esophagus, which was confirmed by
means of subsequent pathologic correlation (Fig 4, E, F). Optical imaging

demonstrated significantly lower relative photon signal in the combination
therapy group (0.56 3 107 photons per
second per square millimeter 6 0.11)
compared with that in the chemotherapy-only group (1.23 3 107 photons

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per second per square millimeter 6
0.41, P , .05), RF hyperthermia–only
group (3.51 3 107 photons per second per square millimeter 6 0.51; P
, .0001), and PBS group (3.91 3 107
photons per second per square millimeter 6 0.73, P , .0001) (Fig 5a, 5c).
We also performed US imaging to evaluate changes in tumor size before and
after treatments (Fig 5b). We detected

the smaller relative tumor volume in
the combination therapy group (0.68 6
0.13) compared with the chemotherapy-only group (1.31 6 0.16, P = .0039),
RF hyperthermia-only group (2.98 6
0.37, P , .0001), and PBS group (3.11
6 0.64, P , .0001) (Fig 5d). Gross
specimens obtained at the end of the
experiment revealed the smallest tumor
size in the combination therapy group

Figure 3

Shi et al

compared with those in the other three
groups. Histologic analysis of apoptosis
by means of deoxyuridine triphosphate
nick-end labeling assay staining further
confirmed more apoptotic cells in the
combination therapy group (41.8% 6
13.8) than in the groups treated with
RF hyperthermia only (5.7% 6 2.3, P
, .001) or chemotherapy only (21.0%
6 7.7, P = .0092) (Fig 5e).

Discussion

Figure 3: Upper panel photographs of representative tumors harvested from four different groups of mice
show smallest tumor size in combination therapy group (Chemo+RFH ) compared with the others. Bottom
panel photomicrographs (magnification, 320) show that apoptosis analysis with deoxyuridine triphosphate
nick-end staining further confirmed more apoptotic cells (blue dots) in combination therapy group than in
other three groups. RFH = RF heat.

The findings of our study showed that
intraesophageal RF hyperthermia can
significantly enhance the direct intratumoral chemotherapy of human
esophageal squamous cancers, as manifested in decreased survival of esophageal squamous cancer cells in in vitro
experiments and shrunken tumor volumes and decreased optical signal intensity values in both in vivo confirmation and validation experiments. Our
study results also provide evidence
that (a) both chemotherapeutic agents
and RF hyperthermia can be delivered
locally to esophageal tumors by means
of an intraesophageal approach, (b) intraesophageal RF hyperthermia can be
used to enhance the direct intratumoral chemotherapy of esophageal cancers, and (c) both optical imaging and

Figure 4

Figure 4: A, Photograph shows microintraesophageal agent–delivery and RF-heating system. B, US image shows guided positioning of microagent delivery system
(arrow) and then intraesophageal inoculation of the target esophageal region with luciferase-labeled human esophageal carcinoma cells through the tip-curved
needle (arrowhead). C, US image shows subsequent injection of cell pellet, which appears as an inhomogeneous hyperechoic signal intensity (arrow). D, Optical
image shows bioluminescence signal from the generated esophageal tumor. E, Gross specimen displays tumor (arrow) adhering to esophageal wall (arrowheads). F,
Photomicrograph (hematoxylin and eosin–stain; magnification, 34) further confirms esophageal carcinoma (EC) with tumor invasion (arrows). RFH = RF heat.
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Figure 5

Figure 5: (a) Optical and x-ray images and (b) US images show tumor response as assessed according to signal intensity (yellow-red colors on a) and tumor size
(arrows on b) in the four groups. There was a larger decrease in both signal intensity and tumor size in the combination therapy group compared with the other three
groups. Graphs show quantitative analysis, which further confirms the signifi­cant decrease in both photon intensity according to (c) optical imaging and (d) relative
tumor volume according to US imaging after combination therapy (chemotherapy and RF hyperthermia) compared with the other groups. (e) Photographs of representative tumors harvested from four different animal groups show smallest tumor (arrows) size in the combination therapy group compared with the others (left and middle
columns, arrowheads indicate esophageal wall). Photomicrographs of apoptosis analysis further confirmed more apoptotic cells (blue dots) in the combination therapy
group than in the other three groups (right column, magnification, 320). RFH = RF heat.

US imaging offer useful tools to assess
the response of esophageal cancers to
chemotherapeutic regimens. This new
8

technique should provide useful diagnostic and therapeutic imaging-guided
methods for further laboratory study

of esophageal malignancies, and further study may allow its translationto
clinical practice.

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EXPERIMENTAL STUDIES: Hyperthermia-enhanced Direct Esophageal Tumor Chemotherapy

Over the past several decades, numerous devices and techniques have
been developed for generation of external hyperthermia (22), including
electromagnetic energy–based RF or
microwave energy and high-intensity
focused ultrasound. However, penetration depth of electromagnetic energy
from a single applicator is limited to a
few centimeters, particularly with microwave energy. In addition, adipose
tissue at the fat-muscle interface can
be overheated easily because of large
reflections of electromagnetic energy
(23). High-intensity focused ultrasound
can result in high absorption by bone
and can cause overheating, while reflection of high intensity focused ultrasound energy at bone and tissue interfaces can negatively affect energy focus
at the target sites (24). Electromagnetic
and high-intensity focused ultrasoundbased approaches are well suited to
treat tumors in large and superficially
located organs such as the liver, lung,
and kidney. These organs are readily
accessible by using standard percutaneous interventional approaches (25,26).
However, precise and effective delivery
of local hyperthermia with electromagnetic energy and high-intensity focused
ultrasound to deep-seated luminal malignancies such as esophageal cancers
still remains a technical challenge in
current clinical practice.
To overcome these clinical limitations of electromagnetic and highintensity focused ultrasound-based
techniques, in this study we specifically
used the MR imaging heating guidewire to deliver thermal energy locally
to esophageal cancers through an intraluminal approach. For the specific
purpose of treating esophageal malignancies, the generation of intraesophageal RF hyperthermia with the MR imaging heating guidewire offers superior
advantages over external hyperthermia
with electromagnetic energy or highintensity focused ultrasound. The thermal energy distribution pattern induced
by the MR imaging heating guidewire
is cylindrically symmetric and homogeneous, and thermal energy can be
delivered within a distance of approximately 1–2 cm around the target site
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n

(21). This capability is well suited to
deliver localized RF heat into the tumor
alone, without causing thermal injury to
adjacent vital organs (such as the spinal
cord) and heat absorption by fat tissue
and bones.
To assess the transferability of this
methodology to humans, experiments
in mid- to large-sized animal models
would be most appropriate. However,
to our knowledge, no such models for
the study of esophageal cancer have
been described. We created a rat
model of molecular imaging–detectable orthotopic esophageal cancer that
meets the requirement that a specific
animal tumor model not only simulates
the pathophysiologic properties of human esophageal cancer but also reveals
the appropriate interactions between
cancer cells and host organs (27,28).
This rat model of orthotopic esophageal
cancer will be a useful tool for further
laboratory investigations into esophageal malignancy. Its advantages include
(a) high reproducibility and cost effectiveness, allowing precise, targeted tumor inoculation with US imaging guidance; (b) relatively rapid tumor growth,
enabling the evaluation of therapeutic
efficacy in a short period of time; (c)
suitability for use with intraesophageal
interventional approaches; and (d) possibility of using both optical imaging
and US imaging to assess the response
of tumors to various treatments.
We designed and manufactured a
microintraesophageal agent delivery
and RF heating system that can be positioned precisely at the target site in
the esophagus with real-time US imaging guidance. By using this microinterventional system, we could accomplish
precisely targeted injection of esophageal squamous cancer cells into the
esophageal wall to create a rat model of
esophageal cancer. The system also allowed local delivery of chemotherapeutic
agents and RF hyperthermia to enhance
the chemotherapeutic effect in esophageal cancers. Because we prelabeled the
esophageal squamous cancer cells with
luciferase and red fluorescence protein
genes, tumor response to RF hyperthermia–enhanced chemotherapy could be
assessed by means of optical imaging,

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Shi et al

which is a sensitive molecular imaging
tool for noninvasive evaluation of optical
signal changes that reflect response of
tumors during treatment.
Our study had limitations. We need
additional experiments to optimize the
temperature and heating duration to
maximize the enhancing effect of RF
hyperthermia on chemotherapy. In addition, we placed a fiber optical thermal sensor to monitor the real-time
temperature change in the esophagus
lumen, which lacks the capability to
reflect the temperature in the relative
deep tissue, such as the spinal cord.
In future studies we will investigate
the feasibility of using real-time MR
thermometry to monitor the temperature changes in the RF heated areas to
warrant the safe use of this technique.
Moreover, since our study was focused
on establishing the “proof-of-principle”
of the technique, we did not include a
control group with systemic chemotherapy to highlight the value of local RF
hyperthermia-enhanced chemotherapy.
In future studies, we will perform a
quantitative analysis of chemotherapeutic drug deposit in esophageal cancers
under different circumstances of drug
delivery approaches, including local intratumoral injection, systemic administration, and RF hyperthermia-enhanced
drug delivery. In addition, long-term tumor follow-up, rather than 14-day follow-up is necessary to investigate the
effect of RF hyperthermia–enhanced
chemotherapy on tumor mass–caused
dysphagia.
In conclusion, intraesophageal hyperthermia can enhance direct intratumoral chemotherapy on rat orthotopic
esophageal cancers, which may open
new avenues for effective management
of esophageal malignancies by means of
simultaneous integration of RF technology, interventional oncology, and direct
intratumoral chemotherapy.
Disclosures of Conflicts of Interest: Y.S. disclosed no relevant relationships. F.Z. disclosed
no relevant relationships. Z.B. disclosed no relevant relationships. J.W. disclosed no relevant
relationships. L.Q. disclosed no relevant relationships. Y.L. disclosed no relevant relationships. Y.M. disclosed no relevant relationships.
K.V. disclosed no relevant relationships. X.Y.
disclosed no relevant relationships.

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EXPERIMENTAL STUDIES: Hyperthermia-enhanced Direct Esophageal Tumor Chemotherapy

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