In Vitro Survival of Nonsmall Cell Lung Cancer Cells Following

Combined Treatment with Ionizing Radiation and Photofrin-mediated

Photodynamic Therapy
Prachi Sharma
1
, Thomas Farrell
2,3
, Michael S. Patterson
2,3
, Gurmit Singh
3,4
, James R. Wright
3,5
,
Ranjan Sur
3,5
and Andrew J. Rainbow*
1
1
Department of Biology, McMaster University, Hamilton, ON, Canada
2
Department of Medical Physics and Applied Radiation Sciences, McMaster University, Hamilton, ON, Canada
3
Juravinski Cancer Centre, Hamilton, ON, Canada
4
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada
5
Department of Oncology, McMaster University, Hamilton, ON, Canada
Received 7 April 2008, accepted 15 May 2008, DOI: 10.1111 j.1751-1097.2008.00401.x
ABSTRACT
It has been suggested that combination high dose rate (HDR)
intraluminal brachytherapy and photodynamic therapy (PDT) in
nonsmall cell lung cancer (NSCLC) may improve efcacy of
treatment, reduce toxicity and enhance quality of life for
patients. To provide a cellular basis for this we examined the
in vitro sensitivity of MRC5 normal lung broblasts and four
NSCLC cell lines following HDR radiation, PDT and combined
HDR radiation and PDT. HDR radiation was cobalt-60 gamma
rays (1.51.9 Gy min
)1
). For PDT treatment, cells were exposed
to 2.5 lg mL
)1
Photofrin for 1824 h followed by light exposure
(20 mW cm
)2
). For combined treatment cells were exposed to
Photofrin and then either exposed to light and 1530 min later
exposed to HDR radiation or exposed to HDR radiation and 15
30 min later exposed to light. D
37
values calculated from
clonogenic survival curves indicated a six-fold difference in
HDR radiation sensitivity and an eight-fold difference in PDT
sensitivity. The effect of combined treatment was not signi-
cantly different from an additive effect of the individual
treatment modalities for the NSCLC cells, but was signicantly
less than additive for the MRC5 cells. These results suggest an
equivalent tumor cell kill may be possible at reduced systemic
effects to patients.
INTRODUCTION
The two main types of lung cancer are small cell lung cancer
and nonsmall cell lung cancer (NSCLC), which are diagnosed
based on how the cells look under a microscope (as reviewed in
[1]). About 85% of all lung cancers are of the NSCLC type.
Lung cancer is the leading cause of cancer deaths in the world
and only approximately 14% of all people who develop lung
cancer survive for longer than 5 years (2). Thus, newer
strategies are needed to improve outcomes in NSCLC.
Lung cancer, once diagnosed, is usually treated with a
combination of surgery, chemotherapy and radiotherapy. It is
well known that tumors from the same histologic group and
stage of development are highly heterogeneous in their
sensitivity to therapy (3). Resistance of tumor cells to
treatment often accounts for the failure of traditional forms
of anticancer therapy. High dose rate intraluminal brachy-
therapy (HDRILBT) and photodynamic therapy (PDT) are
two treatment options for lung cancer that are also used for
palliation of symptoms in many institutions. Endobronchial
tumors are well suited to treatment with either PDT or HDR
brachytherapy and good palliative results have been reported
with NSCLC (46).
Photodynamic therapy involves the use of a photosensitizer,
such as Photofrin, the application of visible light of the
wavelength specic for the photosensitizer and the presence of
oxygen leading to reactive oxygen species-mediated cytotoxic-
ity to the treated cell (7,8). PDT kills tumor cells via apoptosis
and or necrosis both in vivo and in vitro. The particular mode
of cell death in response to PDT depends on experimental
conditions, such as the dose of PDT and the subcellular
localization of the photosensitizer (9). Photofrin, the photo-
sensitizer used in this study, is a partially puried derivative of
hematoporphyrin that is activated by light at 630 nm. Photo-
frin is an approved photosensitizer by the U.S. Food and Drug
Administration. HDRILBT is a form of radiation treatment
given by placing a radioactive isotope in and around a tumor.
Exposure to ionizing radiation leads to rapid necrosis of tumor
tissues principally by nuclear DNA damage (10).
It has been reported previously that the PDT-resistant
murine brosarcoma cell line RIF-8A (11) showed increased
sensitivity to ionizing radiation compared with PDT-sensitive
RIF-1 cells (12). It has also been reported that the radiation-
resistant L5178Y murine lymphoma cell line was more
sensitive to chloroaluminium phtathocyanine-mediated PDT
compared with the radiosensitive LY-S cell line from which it
was derived (13). These results indicate that some radiation-
resistant tumor cells are sensitive to PDT and some PDT-
resistant tumor cells are more sensitive to ionizing radiation. In
addition, there are reports indicating that some photosensiti-
zers can act as radiosensitizers (14,15). These reports
suggest that a combined treatment of tumors with both
*Corresponding author email: rainbow@mcmaster.ca (Andrew J. Rainbow)
2008 The Authors. Journal Compilation. The AmericanSocietyof Photobiology 0031-8655/09
Photochemistry and Photobiology, 2009, 85: 99106
99
Photofrin-mediated PDT and ionizing radiation could be
superior to the use of the single modalities of PDT and
ionizing radiation alone. The in vitro sensitivity of NSCLC cell
lines to the single modality treatment of gamma rays (16) and
PDT (17) has been reported previously. However, there are no
previous reports concerning the sensitivity of NSCLC cell lines
to combined treatment with both Photofrin-mediated PDT
and ionizing radiation.
Adams et al. showed in vivo resistance to Photofrin-
mediated PDT in radiation-induced brosarcoma cells RIF-
8A that were resistant to in vitro Photofrin-mediated PDT (18).
This suggests that the in vitro sensitivity of cells to Photofrin-
mediated PDT could give valuable information on the
response to PDT of cancer cells in vivo. In the present study
we examined the sensitivity of four different NSCLC cell lines
A549, NCIH460, NCIH23 and SKMES1 and a normal lung
broblast cell line (MRC5) to ionizing radiation alone,
ionizing radiation with Photofrin but no light, Photofrin
alone, Photofrin-mediated PDT alone and a combination of
ionizing radiation and PDT using a colony-forming assay.
We report that the combined treatment with HDR radia-
tion and PDT was not signicantly different from an additive
effect of the individual treatment modalities for in vitro
survival of the four NSCLC cells but was signicantly less
than additive for the MRC5 cells. These results suggest that for
some tumor cell types, the combined treatment with HDR
radiation and PDT compared with the individual treatment
modalities may have the potential advantage of doing less
damage to normal lung cells for the same tumor cell kill.
MATERIALS AND METHODS
Cell lines. The NSCLC cell lines A549, SKMES1, NCIH460 and
NCIH23 and the normal human broblast strain MRC5 were obtained
fromATCC(Rockville, MD). A549 is human adenocarcinoma and was
initiated from a human alveolar cell carcinoma. NCIH460 is a human
large cell carcinoma cell line, NCIH23 is a human adenocarcinoma cell
line, SKMES1 is a human lung squamous cell carcinoma line and MRC5
is normal lung broblast tissue derived from a 14-week-old male fetus.
All cell cultures were grown as monolayers in GIBCO RPMI
medium 1640 modied with L L-glutamine, ribonucleosides and deoxy-
ribonucleosides supplemented with 10% fetal bovine serum and
1% antibiotic-antimycotic (100 lg mL
)1
penicillin G sodium,
100 lg mL
)1
streptomycin sulfate and 250 ng mL
)1
amphotericin B
in 0.85% saline) obtained from Gibco-BRL. Cultures were maintained
in 37C humidied air containing 5% CO
2
at 90% humidity. For PDT,
Photofrin + gamma and combination experiments, growth medium
was supplemented with 2% antibiotic-antimycotic to avoid contam-
ination as experiments were performed under minimal light conditions.
Photosensitizer. Photofrin

was obtained from Axcan Pharma Inc.

(QC, Canada). It was reconstituted in 5% dextrose to a concentration
of 2.5 mg mL
)1
. Aliquots (1 and 0.5 mL) of Photofrin were then
prepared in cryovials and stored covered in aluminum foil in a freezer
at )20C in the dark. Storage, dilution steps and incubation period
were performed under experimental conditions avoiding the exposure
of Photofrin to light.
Irradiation sources. The light source was an LED that emits red
light in the visible region of the spectrum at a wavelength range of 620
640 nm. The power output of the source was 20 mJ cm
)2
s
)1
. The
Cobalt-60 gamma ray source was a radiation unit at the Juravinski
Cancer Centre (Hamilton, ON), emitting gamma rays at a dose rate of
140190 cGy min
)1
.
Colony-forming assay: treatment with gamma rays. For low density
seeding of cells, conuent 75 cm
2
asks of cells were trypsinized, cells
were counted using a hemocytometer and the desired number (300
1000 cells per well) was seeded into 6-well plates in 2 mL of growth
medium. Following a 24 h incubation period, the growth medium was
replaced by 2 mL of PBS and the plates were exposed to gamma rays
or mock irradiated. The PBS was then replaced by 2 mL of growth
medium and plates were incubated for 1012 days. Plates were then
xed and stained with approximately 1 mL of crystal violet solution
(63% absolute ethanol, 27% H
2
O, 10% methanol, 5 g L
)1
crystal
violet) and colonies containing at least 32 cells were counted.
Flow cytometry: Photofrin uptake. For the Photofrin uptake exper-
iments, cells were counted on a hemocytometer and seeded for
conuence (1 10
6
cells per well) in a 6-well plate in 2 mL of growth
medium and allowed to incubate for 6 h at 37C and 5% CO
2
. The
medium was then aspirated from each well and replaced with 3 mL of
growth medium containing the desired concentration of Photofrin.
Plates were incubated again, this time for an overnight period (20
24 h) and kept under aluminum foil to minimize the effect of ambient
lighting. Following the 2024 h incubation period, the medium was
aspirated from each well and the cells were washed with 1 mL of
warmed PBS. The PBS was then aspirated from the wells and replaced
with 1 mL of 2 trypsin-EDTA (0.5% trypsin, 5.3 mM M EDTA 4Na).
Cells were trypsinized, 5 mL of growth medium was added and the
solution was mixed several times with a micropipette. The contents of
the well were removed to a 15 mL Falcon tube and centrifuged. The
pellet obtained was resuspended in 1 mL of PBS. The Photofrin
concentration of the cell was measured by ow cytometry using an
excitation wavelength of 488 nm and emission measurements at 620
675 nm. Fluorescence per cell was plotted against Photofrin concen-
tration.
Colony-forming assays: Photofrin-light (PDT) Photofrin-gamma
rays. Photofrin and light (PDT): The sensitivity of cells to PDT was
examined using a high cell density protocol in order to more closely
simulate the clinical conditions of PDT. Conuent 75 cm
2
asks of
cells were trypsinized, counted with a hemocytometer, and 1 10
5
cells well (high cell density) were plated in a 24-well tissue culture
plate. Cells were left to adhere for a minimum of 6 h before treating
with Photofrin. After this time, the medium was aspirated from the
plate, 2 mL of the appropriate Photofrin dilution (2.5 lg mL
)1
) was
overlayed on each well, and plates were incubated again for an
overnight period (1824 h) and kept under aluminum foil to minimize
ambient lighting. After 1824 h of incubation, the Photofrin-contain-
ing medium was aspirated and 2 mL of fresh growth medium without
Photofrin was added to each well. Photofrin-treated plates were
exposed to various doses of visible light using an LCD light source.
Three hours after irradiation, plates were trypsinized, diluted and an
appropriate number of cells were seeded in 6-well plates. After 6
10 days of incubation, the medium was aspirated and colonies were
stained with crystal violet and counted.
Photofrin and gamma rays: For an examination of the effect of
Photofrin on the sensitivity of cells to gamma rays, a high cell density
protocol was used. Conuent 75 cm
2
asks of cells were trypsinized,
counted using a hemocytometer and 1 10
5
cells well were plated in a
24-well tissue culture plate. Cells were left to adhere for a minimum of
6 h before treating with Photofrin. The growth medium was then
aspirated from the plate and replaced with 2 mL of growth medium
containing an appropriate concentration of Photofrin. After 1824 h
of incubation, the Photofrin-containing medium was aspirated and
2 mL of fresh growth medium without Photofrin was added to each
well. Photofrin-treated plates were exposed to 7 Gy or left untreated.
Three hours after irradiation, plates were trypsinized, diluted and an
appropriate number of cells was seeded into 6-well plates and
incubated. After 610 days of incubation, the medium was aspirated
and colonies were stained with crystal violet and counted.
Colony-forming assay for combined treatment with Photofrin-
mediated PDT and ionizing radiation. Combined treatment consisted
of 2.5 lg mL
)1
Photofrin (all NSCLC cell lines), light exposure of
400 s and 7 Gy gamma rays (SKMES1, A549 and NCIH460), light
exposure of 100 s and 2 Gy gamma rays (NCIH23) or light exposure
of 100 s and 7 Gy gamma rays (MRC5). Conuent 75 cm
2
asks of
cells were trypsinized, counted using a hemocytometer and 1 10
6
cells well (high cell density) were plated in a 12-well tissue culture
plate. Cells were left to adhere for a minimum of 6 h before treating
with Photofrin. The medium was then aspirated from each well and
replaced with 2 mL of growth medium containing an appropriate
concentration of Photofrin. Plates were incubated again for 1824 h
and kept under aluminum foil to minimize ambient lighting. The
Photofrin-containing medium was then aspirated and 2 mL of fresh
100 Prachi Sharma et al.
growth medium without Photofrin was added to each well. In order to
determine whether the order of combined treatment inuences tumor
cell kill, we used the following experimental protocol. After adding
fresh growth medium without Photofrin, cells were either (1) rst
exposed to light and within 1530 min exposed to gamma rays or (2)
exposed to gamma rays and within 1530 min exposed to light. Cells
were then incubated for 3 h covered in aluminum foil in the dark at
37C-humidied air containing 5% CO2 at 90% humidity. After the
3 h incubation, the cells were trypsinized, diluted and an appropriate
number of cells was seeded in 6-well plates. After 610 days of
incubation, the medium was aspirated and colonies were stained with
crystal violet and counted. Experimental sets used in the combination
experiments included: (1) no drug no light (NDNL); (2) drug
alone (DNL); (3) light alone (NDL); (4) PDT; (5) gamma rays;
(6) Photofrin + light + gamma rays; (7) Photofrin + gamma rays +
light.
RESULTS
Sensitivity of nonsmall cell lung cancer cell lines to gamma rays
Figure 1 shows pooled data obtained from colony survival
assays using cobalt-60 gamma rays. It can be seen that A549
was the most resistant cell line and NCIH23 was the most
sensitive to ionizing radiation and all other cell lines NCIH460,
SKMES1 and MRC5 showed intermediate sensitivities. For
each individual survival curve obtained in independent exper-
iments, the dose required to reduce the surviving fraction of
cells to 37%, or D
37
value, was extrapolated. The average D
37
values for colony survival following gamma ray treatment for
a number of experiments on each cell line are shown in
Table 1, column 2. The D
37
values indicated a six-fold
difference in sensitivity to gamma rays with A549 being the
most resistant cell line and NCIH23 being the most sensitive.
The plating efciency of the different cell lines is shown in
Table 1, column 4.
Uptake of Photofrin by nonsmall cell lung cancer cell lines
Results for Photofrin uptake are shown in Fig. 2. It can be
seen that all the cell lines showed a similar Photofrin uptake
per cell over the range of Photofrin concentrations employed.
Sensitivity of nonsmall cell lung cancer cell lines to
Photofrin-mediated PDT
We rst examined the effect of different Photofrin concentra-
tions without light exposure on cell survival, for the normal
lung broblasts MRC5 and the four NSCLC cell lines (A549,
SKMES1, NCIH23 and NCIH460). For an incubation time of
1824 h, 10 lg mL
)1
of Photofrin alone signicantly reduced
survival for MRC5 cells but not for any of the NSCLC cell
lines, whereas 2.5 and 5 lg mL
)1
of Photofrin alone had no
signicant effect on colony survival for MRC5 or any of the
NSCLC cell lines when compared with control (NDNL) plates
(data not shown).
The clinical recommended dose for Photofrin is 2 mg kg
)1
given 2448 h prior to light exposure treatment at the tumor
site. In order to closely simulate these clinical conditions of
PDT we used a Photofrin concentration of 2.5 lg mL
)1
for
an incubation time of 1824 h followed by varying light
exposure for the in vitro sensitivity studies. Figure 3 shows
the effect of PDT on the survival of the four NSCLC cell
lines (A549, NCIH23, NCIH460 and SKMES1) and the
normal lung broblasts MRC5. It can be seen that A549
was the most resistant cell line and NCIH23 was the most
sensitive to Photofrin-mediated PDT and all other cell
linesNCIH460, SKMES1 and MRC5showed intermedi-
ate sensitivities. For each individual survival curve obtained
in independent experiments, the light exposure required to
reduce the surviving fraction of cells to 37%, or D
37
value,
was extrapolated. The average D
37
values for colony survival
following Photofrin-mediated PDT for a number of exper-
iments on each cell line are shown in Table 1, column 3.
The D
37
values calculated from the survival curves indicated
Figure 1. Clonogenic survival of selected nonsmall cell lung cancer cell
lines and a normal lung broblast following treatment with varying
doses of gamma rays. Cells were seeded at low density, treated with
varying doses of gamma rays and assayed for clonogenic survival 8
12 days later. Data points are mean values standard error from two
to four independent experiments each performed in triplicate.
Table 1. D
37
values obtained from colony survival assays for exposure
to gamma rays and Photofrin plus visible light treatment to NSCLC
cells and normal lung broblasts.
Cell lines
Average
D
37
SE
(Gy)
Average
D
37
SE
(light exposure in s)
Average
plating
efciency SE
MRC5 1.42 0.16 (4) 64 23 (4) 7.5 2.5 (5)
NCIH23 0.85 0.05 (3)* 62 8 (3) 10.4 0.9 (9)
SKMES1 3.72 0.15 (2)* 222 64 (3)* 21.9 7.2 (6)
NCIH460 4.89 0.11 (2)* 223 13 (4)* 55.9 17.0 (4)
A549 5.46 0.44 (4)* 478 81 (4)* 46.2 11.0 (6)
NSCLC = nonsmall cell lung cancer. The average D
37
SE are
reported for gamma rays (column 2) and PDT (column 3). Average
plating efciencies are shown in column 4. The number of experiments
used to determine each of the values is shown in parentheses. *Values
that are signicantly different from that for the MRC5 broblasts by a
two-sample independent t-test (P < 0.05).
Photochemistry and Photobiology, 2009, 85 101
an eight-fold difference in sensitivity to PDT with A549
being the most resistant cell line and NCIH23 being the
most sensitive.
Radiosensitization of nonsmall cell lung cancer cell
lines by Photofrin
In order to study the possible radiosensitizing properties of
Photofrin for NSCLC cells, we examined the effect of
increasing Photofrin concentrations (2, 5 and 10 lg mL
)1
)
on clonogenic survival following a dose of 7 Gy. 7 Gy was
used as it is a typical dose of ionizing radiation given during
brachytherapy treatment of NSCLC. We found no signicant
difference in survival following 7 Gy for the normal MRC5
cells or any of the NSCLC cell lines when cells where incubated
for 1824 h with Photofrin concentrations of 2 or 5 lg mL
)1
followed by gamma rays, as determined by a two-sample
independent two-tailed t-test (data not shown). As the clinical
recommended dose for Photofrin is 2 mg kg
)1
, this suggests
there would be little if any radiosensitizing effect of Photofrin
in the clinical situation. At 10 lg mL
)1
of Photofrin we did
detect a radiosensitizing effect in the MRC5 cells but not in
any of the NSCLC cells, as determined by a two-sample
independent one-tailed t-test but not in a two-sample inde-
pendent two-tailed t-test (data not shown). Our results suggest
a radiosensitizing effect of Photofrin for MRC5 cells at
10 lg mL
)1
, but not 2 or 5 lg mL
)1
of Photofrin and no
radiosensitizing effect for any of the NSCLC cells at any of the
concentrations tested.
Sensitivity of nonsmall cell lung cancer cell lines to combined
Photofrin-mediated PDT and ionizing radiation
In our study both PDT and HDR gamma rays were given over
a period of not more than 30 min because we felt it would be
more convenient and less traumatic for the patient if both
treatments were carried out during a single endoscopy proce-
dure. The two major questions addressed were (1) is there any
effect of order of combined treatment on NSCLC cell lines and
normal lung broblast MRC5 and (2) is combined treatment
of HDR ionizing radiation and PDT more effective than
individual treatment?
Survival of cells in all the experimental sets was calculated
relative to the control set (NDNL). Table 2 shows the effect of
order of combined treatment on NSCLC cell lines. Mean
surviving fractions SE (calculated from all experiments) of
NSCLC cells and normal lung broblast MRC5 are reported
when cells were exposed to Photofrin and then either exposed
to light and 1530 min later exposed to gamma radiation (a) or
exposed to gamma radiation and 1530 min later exposed to
light (b). The ratio of (b) (a) for all the experiments was also
calculated as mean values SE. Results show that although
light followed by gamma rays resulted in a somewhat greater
tumor cell kill compared with gamma rays followed by light,
this difference was not signicant for any of the cell lines
tested. However, this difference was signicant (as determined
by a one-sample one-tailed t-test) when data for all NSCLC
cell lines were pooled.
Figure 3. Effect of Photofrin and light on the survival of NSCLC cell
lines and MRS5 normal lung broblasts using a high cell density
protocol. Cells were incubated with 2.5 lg mL
)1
Photofrin for 1824 h
and subsequently exposed to varying visible light exposures at a power
output of 20 mJ cm
)2
s
)1
. Three hours after exposure to light, cell
monolayers were trypsinized, diluted and plated on 6-well plates.
Colonies were stained and counted after 7 days. Data points are mean
values standard error of three to four independent experiments
each performed in triplicate.
Figure 2. Uptake of Photofrin per cell for the NSCLC cell lines and
MRC5 normal lung broblasts. Cells were incubated in humidied air
at 37C and 5% CO
2
with varying concentrations of Photofrin
overnight (2024 h). The Photofrin concentration of cells was mea-
sured by ow cytometry using an excitation wavelength of 488 nm and
emission measurements at 620675 nm. Fluorescence per cell was
plotted against Photofrin concentration. Data points are mean
values standard error from two to three independent experiments
each performed in duplicate.
102 Prachi Sharma et al.
Table 3 shows the effect of combined treatment in compar-
ison with predicted survival. The survival of NSCLC cells
following combined treatments (a) and (b) was compared with
predicted survival values based on survival following PDT and
gamma rays alone (c) (PDT X gamma rays). As P + L then
gamma (a) was not signicantly different from P + G then
light (b) for each of the cell lines, we were able to pool the data
from both the treatments to compare with the expected
survival.
Survival of cells following the combined treatment was
greater than that expected based on an additive effect for
SKMES1, NCIH460, NCIH23 and MRC5. This less than
additive effect of the combined treatment was signicant for
the MRC5 cells in a two-sample independent t-test. In
contrast, the less than additive effect in the SKMES1 and
NCIH23 cells was only signicant in a one-tailed t-test, and
was not signicant in either test for the NCIH460 cells. By
comparison, the survival of A549 following combined treat-
ment was less than that predicted based on an additive effect,
suggesting a synergistic effect of combined treatment in A549
cells. However, this synergistic effect in A549 cells was only
signicant in one-sample one-tailed t-test, but not by one-
sample two-tailed t-test or a two-sample independent t-test.
Thus, as determined by the more stringent two-sample
independent t-test, the combined treatment with HDR radia-
tion and PDT was not signicantly different from an additive
effect of the individual treatment modalities for in vitro
survival of the four NSCLC cells. In contrast, the combined
treatment was less than additive for the MRC5 cells. This
suggests that for some tumor cell types, the combined
treatment with HDR radiation and PDT, when compared
with the individual treatment modalities, may have the
potential advantage of doing more damage to the tumor cells
than to normal lung cells.
DISCUSSION
Of the four NSCLC cell lines examined, A549 was the most
resistant to gamma rays, NCIH23 was the most sensitive and
all other cell lines NCIH460, SKMES1 and MRC5 showed
intermediate sensitivities. These results are consistent with
those of Carmichael et al., who examined the X-ray sensitivity
of several NSCLC cell lines and reported A549 as the most
resistant, NCIH23 the most sensitive with NCIH460 having
intermediate sensitivity following a dose of 2 Gy (16).
We report a similar Photofrin uptake per cell over the range
of Photofrin concentrations employed for all the NSCLC cells
and the normal MRC5 cells tested. In vitro cellular uptake and
retention of Photofrin has been found to be a passive process
not involving energy expenditure. pH and temperature of the
incubation media have been found to profoundly inuence
these processes, while a complex relationship exists between
the physiologic state of the cell and accumulation of the
photosensitizer (19). Consistent with the results of the present
study, Perry et al. also showed no signicant difference in
Photofrin uptake between A549 and NCIH460 cells (17).
Although some studies have reported that malignant cells take
up more drug in comparison with normal cell lines (as
reviewed in Oleinick et al. [20]), other studies have reported
that normal human broblast cells show greater uptake
Table 2. Effect of order of combined treatment on NSCLC cell lines.
Cell lines No. Exp.
Surviving fraction
(a) P + L then gamma
Surviving fraction
(b) P + G then light Ratio (b) (a) P-value*
SKMES1 3 0.027 0.02 0.027 0.019 1.02 0.022 0.240
A549 4 0.056 0.04 0.073 0.036 2.29 0.88 0.140
NCIH460 4 0.0036 0.0019 0.0043 0.002 2.45 1.30 0.180
NCIH23 3 0.057 0.022 0.069 0.027 1.27 0.095 0.053
MRC5 3 0.026 0.003 0.032 0.009 1.27 0.38 0.27
NSCLC = nonsmall cell lung cancer. Mean surviving fractions SE of NSCLC cells and normal lung broblasts MRC5 are shown for cells
exposed to Photofrin and then either (a) exposed to light and 1530 min later exposed to gamma radiation or (b) exposed to gamma radiation and
1530 min later exposed to light. Ratio of (b) (a) for all the experiments on a given cell line is shown as the mean ratio SE in addition to the
number of experiments (n) used to determine the values by using a high cell density protocol. *Not signicantly greater than one by one-sample
one-tailed t-test.
Table 3. Effect of combined treatment of PDT and gamma rays on NSCLC cell lines and normal lung broblasts.
Cell lines
No.
Exp.
Surviving fraction
(a) P + L then gamma
Surviving fraction
(b) P + G then light
(c) Expected
survival
Ratio (a) (c)
and (b) (c)
Two-sample ind t-test
P-value Effect
SKMES1 3 0.027 0.020 0.027 0.019 0.014 0.012 2.59 0.42 0.52 Less than additive
A549 4 0.056 0.040 0.073 0.036 0.077 0.037 0.733 0.12 0.77 Synergistic
NCIH460 4 0.0036 0.0019 0.0043 0.002 0.0033 0.0011 1.66 0.53 0.77 Less than additive
NCIH23 3 0.057 0.022 0.069 0.027 0.034 0.014 2.01 0.17 0.28 Less than additive
MRC5 3 0.026 0.003 0.032 0.009 0.006 0.003 12.8 6.4 0.01* Less than additive
PDT = photodynamic therapy; NSCLC = nonsmall cell lung cancer. The survival of NSCLC cells following combined treatments (a) and (b) was
compared with predicted survival values based on survival following PDT and gamma rays alone (c). Ratios obtained from (a) (c) and (b) (c) were
pooled as there was no signicant difference between the two treatments for each of the cell lines. Following combined treatment, the survival of
MRC5 cells, but not NSCLC cells, was signicantly different from that expected (based on an additive effect of PDT plus gamma radiation) by a
two-sample independent t-test*.
Photochemistry and Photobiology, 2009, 85 103
compared with immortalized Li-Fraumeni syndrome cells (21).
In contrast, the results of the current work show no difference
in Photofrin uptake per cell between the MRC5 normal lung
broblast and the four NSCLC cell lines.
In the present report, treatment with 2.5 and 5 lg mL
)1
of
Photofrin alone for 1824 h had no signicant effect on colony
survival for MRC5 or any of the NSCLC cell lines. Perry et al.
showed that there was no effect on survival of six NSCLCs
(A549, NCIH23, NCIH841, NCIH460, JMN and NCIH520)
and a normal lung broblast CCL-210 when cells were
incubated with 25 lg mL
)1
of photofrin alone for 2 h (17).
Mathews et al. showed that there was no effect on survival of
A549 on varying Photofrin concentrations (2.5, 25 or
50 lg mL
)1
) for different periods of time (2, 4 or 6 h) (22).
Of the four NSCLC cell lines tested here, A549 was the most
resistant to Photofrin-mediated PDT, NCIH23 was the most
sensitive and the other NSCLC cell lines, NCIH460 and
SKMES1 showed intermediate sensitivities. Perry et al. have
also reported that NCIH23 was more sensitive to Photofrin-
mediated PDT compared with A549 (17). The PDT sensitivity
of the MRC5 normal lung broblast was signicantly greater
than A549, NCIH460 and SKMES1, but not signicantly
different compared with NCIH23 cells. Perry et al. also
reported increased PDT sensitivity of the CCL-210 normal
lung broblast compared with A549, NCIH23 and NCIH460
cells (17). Taken together, these results suggest that normal
lung broblasts are inherently more sensitive to PDT com-
pared with most NSCLC cells. However, as there are no
published reports on the comparative PDT sensitivity of
normal thoracic organs and cells in clinical PDT, it remains to
be determined whether lung broblasts are an appropriate
normal tissue cell line for the evaluation of clinical response.
Perry et al. reported a comparable in vitro sensitivity for the
NCIH460 NSCLC cell line and the NCIH841 small cell variant
following Photofrin-mediated PDT (17). In contrast, using
tumor xenografts in Balb c nude mice, Chin et al. reported
increased sensitivity of tumor xenografts of NCIH460 NSCLC
cells compared with NCIH526 small cell lung cancer cells
following chlorin e6-polyvinylpyrrolidone-mediated PDT (23).
These latter results suggest that second- and third-generation
photosensitizers, such as chlorophyll chlorin derivatives, may
have distinct advantages as photosensitizers for PDT treat-
ment of NSCLC and clinical studies using mono-L L-aspartyl
chlorin e6 for the treatment of early-stage lung cancer
demonstrated excellent antitumor effects and safety (24,25).
In the current work, PDT sensitivity did not correlate with
photosensitizer uptake. It has been reported that intracellular
localization of photosensitizer may differ between cells or
even substrains of the same cells (2628) suggesting that
differences in sensitivity to PDT of the four NSCLC cell lines
and MRC5 cell line might be due to differences in cellular
organelle uptake by the cell lines. Bohmer and Morstyn
showed that different cell lines under identical conditions
took up different amounts of sensitizer. These differences
were dependent upon the cell size, with larger cells taking up
more sensitizer than small cells (29). The same Photofrin
uptake per cell in all the cell lines in our experiments under
identical experimental conditions suggests that this might be
because of the same size of the various NSCLC cell lines
used in our study. However, it should be noted that sensitizer
uptake measurements were made using the entire population
of cells, while the PDT sensitivity assessments represent only
the proportion of cells that actually form colonies. Ideally,
the appropriate way to correlate sensitizer uptake with PDT
sensitivity would be to use only cell lines with 100% plating
efciencies, a rare characteristic of most human lung cancer
cell lines. Whether the sensitivity of a few percent of cells
that are actually able to form colonies is representative of the
entire population of cells is not clear and has been discussed
at length by Perry et al. (17).
Perry et al. have reported a general association between
PDT sensitivity and the plating efciency of the cell line (17).
In the present work we also show that the cell lines with low
plating efciency (MRC5 and NCIH23) had an increased
sensitivity to gamma rays as well as Photofrin-mediated PDT.
Results from colony survival experiments when plating ef-
ciencies are low are always a concern when such data are
extrapolated to clinical outcome. In addition, differences in
tumor vasculature (30) and the host immune response (31) can
markedly inuence tumor response to PDT. Notwithstanding,
our results demonstrate differences among NSCLC cell lines
and their sensitivity to gamma rays and Photofrin-mediated
PDT under controlled in vitro conditions. Such differences
may be responsible for some of the failures seen in clinical
PDT and brachytherapy.
In vitro radiosensitization by Photofrin has been reported
for Rif-1 murine brosarcoma cells (12), U-373 MG glioblas-
toma cells and RT4 human bladder cancer cells, but not HT29
colon adenocarcinoma cells (32). In the present work we show
a signicant radiosensitizing effect of Photofrin in MRC5 cells
at 10 lg mL
)1
but not at 2 and 5 lg mL
)1
Photofrin and no
radiosensitizing effect of Photofrin in the NSCLC cells for any
of the Photofrin concentrations examined. Taken together,
these results indicate that in vitro radiosensitization by
Photofrin is possible in some, but not all cell types and is
dependent on photosensitizer concentration. Several other
studies using murine tumor models have demonstrated the
in vivo efcacy of Photofrin as both a specic and a selective
radiosensitizing agent (3335). In addition, early follow-up
results of a phase I trial on the clinical application of Photofrin
as a radiosensitizer are encouraging (36). The mechanism of
the radiosensitizing effect is not completely understood. The in
vitro data support the hypothesis that the radiosensitizing
action involves OH-radicals in addition to a potential impair-
ment of repair mechanisms after sublethal damage of ionizing
radiation (37).
The rationale of cancer treatment with a combination of
different therapeutic modalities is to obtain improved tumor
control with minimal damage to normal tissues. There are
reports suggesting that PDT and ionizing radiation act by
independent mechanisms, such that combined in vitro treat-
ment with PDT and gamma rays results in an additive effect
(3840). In contrast, although combined PDT and ionizing
radiation showed an additive effect in Chinese hamster ovary
cells and T24 human bladder carcinoma cells, there was a
synergistic interaction in L929 mouse broblasts (39). These
results, together with the results of the current work, indicate
that the outcome of combined PDT and ionizing radiation
treatment can be additive, less than additive or synergistic
depending on the cell type and the conditions of the combined
treatment. In an in vivo clinical study, Freitag et al. showed
that the combination of PDT and brachytherapy for treating
104 Prachi Sharma et al.
patients with lung cancer and extensive endobronchial tumor is
safe and had excellent therapeutic efcacy. Biopsy specimens
were taken from the treated sites during bronchoscopy
56 weeks after PDT and brachytherapy with iridium-192
was administered. It was found that the combined treatment
had a complete histologic response rate of 97% (3). In the
present in vitro study we report that the combined treatment
with HDR radiation and PDT was not signicantly different
from an additive effect of the individual treatment modalities
for survival of the NSCLC cell lines, but was signicantly less
than additive for the MRC5 cells. This suggests that combined
treatment, compared with the individual treatment modalities,
might have the potential advantage of doing less damage to
normal lung cells for the same tumor cell kill such that an
equivalent tumor cell kill in vivo may be possible at reduced
systemic effects to patients.
AcknowledgementThis research was supported by grants from the
Ontario Institute of Cancer Research.
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