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Experimental Therapeutics |
Department of Radiation Oncology, University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The depletion of oxygen during PDT has been measured with oxygen-sensitive electrodes during illumination of tumor spheroids in vitro (4) and animal tumors in vivo (1 , 5 , 6) . Decreases in microvessel oxygen tensions during illumination have been measured by optical spectroscopy of phosphorescent probes (7) . Treatment conditions creating oxygen depletion are associated with poorer tumor responses, whereas those favoring oxygen maintenance increase tumor cell kill (8, 9, 10) , lead to more PDT-associated vascular damage (5) , and ultimately, improve the cure rate of mouse tumors (11, 12, 13) . The benefits of tumor oxygen maintenance during PDT and the dependence of oxygen consumption on the treatment protocol highlight the need for investigation of PDT-created hypoxia. Sensitive methods of quantifying and evaluating the distribution of tumor oxygen could help in the development and optimization of PDT protocols.
Hypoxia markers, drugs that are bioreduced under hypoxic conditions, are one method of studying tissue oxygenation. Such markers have been used by others to describe the presence of hypoxia in tumors after PDT treatment (14 , 15) . In these studies, increased tissue hypoxia was detected in the hours after PDT, in association with decreases in vascular perfusion. In contrast to hypoxia that develops during illumination, hypoxia after PDT may improve tumor response because damage to vasculature can contribute to tumor cures (16) . Thus, the distinction between tumor hypoxia developing during illumination from that created after treatment is important in the investigation of PDT effects on tumor oxygenation.
This report describes the use of the 2-nitroimidazole hypoxia marker EF3 for detecting tumor hypoxia during or shortly after PDT. EF3 is closely related to the well-characterized marker EF5. It has been shown that EF5 covalently binds to hypoxic cells, forming adducts at a rate that varies inversely with oxygen concentration (17) . The cell-bound hypoxia marker is detected with a fluorochrome-conjugated monoclonal antibody and quantified by flow cytometry or visualized by fluorescence microscopy (18) . Tumor hypoxia, as labeled by EF5, directly correlates with radiobiologically detected tumor hypoxia (19) and predicts radiation resistance (20) . In this study, we demonstrate that EF3 binding detects increases in tumor hypoxia during PDT illumination. Tumor oxygenation during illumination is studied separately from that found before or after treatment.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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medium (Life Technologies,
Inc., Grand Island, NY) supplemented with 10% FCS, 1%
penicillin and streptomycin, and 300 µM
L-glutamine. For in vitro studies,
cells were passaged no more than six times after cell isolation from a
tumor. For in vivo experiments, the RIF tumor was propagated
on the shoulders of female C3H mice (Taconic, Germantown, NY) by
intradermal injection of 3 x 105
cells. Tumors were treated 
1 week after injection, at a size of
5–7 mm in diameter. At this size, tumors were free of visible
necrosis.
Photosensitizer and Hypoxia Marker.
Photofrin was purchased from QLT Phototherapeutics Inc. (Vancouver, BC,
Canada), reconstituted in saline to 2.5 mg/ml, and then frozen in
aliquots. For injection, the drug was further diluted to 0.5 mg/ml, and
mice received 5 mg/kg (via tail vein) 20–24 h before PDT
illumination.
The hypoxia marker EF3 was chosen for these studies because it is a more soluble drug than EF5. These drugs demonstrate a similar oxygen dependence to binding, but the increased solubility of EF3 allows the use of higher injected drug doses for the short incubation times of this study. EF3 was synthesized by Dr. M. Tracy and colleagues (SRI International, Menlo Park, CA) and dissolved in saline (20 mM) for mouse injections (via tail vein) at 10 ml/kg (52 mg/kg). Earlier studies with EF5 demonstrated relatively even drug distribution among body tissues (21) ; therefore, an injection of 10 ml/kg of 20 mM EF3 produced a whole body concentration of 200 µM. 14C-labeled EF3 was synthesized by coupling 2-14C-labeled azomycin acetate (NEN DuPont, Boston, MA) to 3,3,3-trifluoropropylamine.
Light Treatment.
Tumor-bearing, photosensitized (or control) animals were treated with
135 J/cm2, delivered at 75
mW/cm2. These conditions were chosen because a
fluence rate of 75 mW/cm2 was shown to deplete
RIF tumor oxygenation, based on needle electrode studies
(5)
, and the rate permitted the delivery of a curative
fluence (135 J/cm2) over a 30-min period
(11)
.
Illumination was performed using a KTP YAG pumped dye module
(Laserscope, San Jose, CA) tuned to produce 630 nm light. Light was
delivered through micRolens-tipped fibers (Rare Earth Medical,
West Yarmouth, MA) for illumination of a 1-cm diameter treatment area
at 75 mW/cm2. Light intensity was measured with a
power meter (Coherent, Auburn, CA). Mice were treated with one of the
following illumination protocols: (a) Control animals
received EF3 with tumor excision performed under anesthesia (ketamine
at 175 mg/kg plus xylazine at 10 mg/kg i.p.) 30 min later. Control
conditions included EF3 plus Photofrin (no light), EF3 plus
illumination (no Photofrin), EF3 alone (neither Photofrin nor
illumination), and tumor alone (no EF3, Photofrin, or illumination).
For the light controls, illumination was performed during the 30-min
EF3 incubation. (b) Tumor hypoxia during PDT was studied in
animals receiving EF3 within 3 min before illumination, with tumor
excision performed immediately after treatment. Anesthesia was
administered during the last 5 min of treatment without interrupting
illumination. (c) Tumor hypoxia after PDT was studied in
mice receiving EF3 within 3 min after illumination for tumor exposure
to drug over the ensuing 30 min. In each treatment group, some animals
also received (via orbital plexus) bisbenzamide solution (30
mg/kg in saline; Hoechst 33342; Sigma, St. Louis, MO) at 1.5 min
before tumor excision. The resulting fluorescence allowed visualization
of perfused vasculature in frozen sections cut from these tumors. Mice
to receive Hoechst injection during the last 1.5 min of PDT were
anesthetized before illumination was begun. EF3 binding in mice
anesthetized for the entire 30-min incubation was indistinguishable
from that in mice anesthetized only for tumor removal; therefore, mice
receiving the same EF3 treatment were pooled regardless of a short or
30-min anesthesia time.
Antibody Staining.
Excised tumors were cut in half perpendicular to the skin surface. One
tumor half was coated with Tissue-Tek OCT compound, placed on
saline-moistened filter paper, and then frozen on dry ice. These
samples were sectioned at 14 µm (Zeiss Microm HM 505 N
cryostat) for immunohistochemistry and fluorescence microscopy. The
other tumor half was cooled to inhibit further EF3 metabolism and
enzymatically digested (167 units/ml collagenase XI, 250
units/ml DNase I, 0.25 mg/ml Pronase E; all from Sigma) to produce a
single cell suspension. Both the cell suspensions and the tissue
sections were stained for EF3 binding using a previously described
protocol (17)
. Briefly, samples were fixed with 4% PF,
rinsed in Dulbecco’s PBS (Sigma), and blocked in PBS containing 0.3%
Tween 20 and 1.5% albumin, plus 20% nonfat milk and 5% normal mouse
serum. Antibody staining was for 4.5–5 h using a monoclonal antibody
(ELK5-A8) conjugated to the fluorochrome Cy5 (Amersham Life Sciences,
Arlington Heights, IL) for study by flow cytometry, or to Cy3
(Amersham) for study by fluorescence microscopy. Samples were rinsed in
PBS containing 0.3% Tween 20 and then PBS with no Tween 20, and stored
in 1% PF until flow cytometry or fluorescence microscopy <1 week
later. Staining controls included the evaluation of fluorescence in
untreated cells and nonspecific antibody binding in tumors from animals
not treated with EF3.
In Vitro EF3 Incubations.
RIF cells were exposed to EF3 under controlled oxygen concentrations
using a previously described procedure (17)
. Cells
(1 x 106) were plated in Ex-Cell
610-HSF medium (JRH Bioscience, Lenexa, KS) containing 25
mM HEPES (Life Technologies), 10% FCS, and 1% antibiotics
in the center of 60-mm glass dishes that had been treated with alkaline
media (15% 0.5 M carbonate, 15% newborn calf serum, and
70% water) and 0.1% gelatin to promote cell attachment. After
overnight incubation, cell medium was replaced with medium containing
30 µM EF3 or 20 µM EF3 plus 10
µM 14C-EF3. Dishes were placed in
aluminum O-ring-sealed chambers and connected to a manifold to allow
the evacuation of precise partial pressures of air from each chamber
and its replacement with N2. After 30 min of
gas exchanges to reach the desired oxygen concentration (0.005–10%
O2), the chambers were removed from the manifold,
brought to 37°C over 15 min, and gently shaken at this temperature
for 3 h of drug incubation. Cells incubated in nonradioactive EF3
were collected with trypsin, fixed, and stained for flow cytometry as
described above. Cells incubated in 14C-EF3 were
lysed with 5% trichloroacetic acid, and the radioactivity in the
acid-soluble and acid-insoluble components was counted using standard
liquid scintillation techniques and a Packard 1900 TR counter
(22)
. EF3 binding is reported in the acid insoluble
fraction, corresponding to EF3 detected by antibody staining
(17)
.
For studies of the time dependence of EF3 binding, cells in suspension were gently stirred in a spinner flask under a constant flow of N2 gas. EF3 at a final concentration of 175 µM was added to the flask (using a syringe inserted in an opening in a ceramic stopper), and at given time points after EF3 injection, cell aliquots were removed for antibody staining and flow cytometry. The relative oxygen concentration in the flask was monitored with a Clark-style ceramic electrode (23) .
Flow Cytometry.
Flow cytometric analysis was performed with a FACSCalibur (Becton
Dickinson, San Jose, CA) maintained by the Flow Cytometry Facility at
the University of Pennsylvania Cancer Center. Cells to be analyzed were
suspended at 0.5–1 x 106
cells/ml in 1% PF and strained (22 µm filter) to eliminate cellular
aggregates. Before each session, machine settings were adjusted such
that a cellular standard produced the same absolute fluorescence. This
standard was created by treating V79 cells in vitro with a
predetermined exposure of hypoxia marker at a known oxygen
concentration (100 µM for 4 h; <0.005%
O2). The standard was stained for the hypoxia
marker in parallel with the experimental samples, and its Cy5
fluorescence was set at 1000. All samples were read on the FL4 channel
(
ex = 635 nm,
em = 661 nm) with a threshold
forward scatter of 20. Data were plotted as histograms (cell count
versus fluorescence intensity) in Cell Quest (Becton
Dickinson) and read into Excel 5.0 (Microsoft) using FCS Assistant
v1.3.1a ß
(shareware).5
Cumulative frequency data were calculated in Excel 5.0.
Fluorescence Microscopy.
Fluorescence microscopy was performed on a Nikon Lab-Phot microscope
equipped with a 100 W high-pressure mercury arc lamp, cooled (-25°C)
CCD camera ("Quantix," KF1400, Grade I defects;
Photometrics), and automatic stage advancement (99S00 stage with
0.1 µm step size; Ludl Electronic Products). Appropriate
filter cubes for each fluorochrome were purchased from Omega Optical
(Brattleboro, VT); residual infrared light from the
filter cubes was eliminated by two serial XF86 filters (Omega Optical)
at the camera base. The camera and stage were controlled by a Macintosh
9600 Power PC computer, and adjacent microscopic fields of a section
were photographed for later assembly into a single image. Imaging
software (IPLab Spectrum; Scanalytics, Inc., Fairfax, VA) precisely
recorded the position of each photograph, enabling a section to be
rephotographed for different fluorochromes at the same position. Images
of Hoechst-labeled blood vessels were made after fixation, and then
sections were stained for EF3 and rephotographed. A third photograph
was taken after the sections were flooded with Hoechst solution and
rinsed; this technique labeled nuclei within the section, indicating
section placement within the image and allowing identification of
tissue edges. To permit accurate comparisons between sections
photographed on different days, an image of hemocytometer-loaded
calibration dye (Cy3 fluorochrome) was taken with each photography
session and used to assess day-to-day variations in the fluorescence
microscopy light source. Photography and image reconstruction were
performed in IPLab Spectrum. Adobe PhotoShop 3.0 (Adobe Systems, Inc.,
Mountain View, CA) was used to colorize and overlay the three
photographs of each section. All images were taken in grayscale, and
colors were produced by converting the image to red/blue/green color,
and then turning off the green and blue for EF3 versus the
red and blue for Hoechst-labeled vessels. Thus, EF3 appears red and
Hoechst-labeled vessels appear green to aid in visual discrimination in
photos. Color intensity was calculated to account for differences in
the exposure time used for each section. Because images were always
photographed for optimal exposure (not over- or underexposed), sections
demonstrating more EF3 binding were represented by shorter exposure
times, not necessarily a brighter image. Quantitative comparison of the
images used the observed distribution of pixel intensities, modified by
the exposure time and adjusted for the dye standard.
Statistics.
The statistical significance of differences among the flow cytometric
data were calculated by the Wilcoxon test performed by JMP (SAS
Institute Inc., Cary, NC). P 
0.05 was
considered significant.
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RESULTS |
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200 µM EF3. At the conclusion of a 30-min
incubation, plasma drug levels of EF3 were 150
µM (data not shown) as a result of drug
metabolism; therefore, an average in vivo exposure was 175
µM EF3 for 30 min. For in vitro
incubation of severely hypoxic RIF cells in 175
µM EF3, median fluorescence intensity was
linear for exposure times of 1.5–60 min (Fig. 1
). The averaged median fluorescence intensity of severely hypoxic RIF
cells exposed to 175 µM EF3 for 30 min was 240.
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shows the median fluorescence intensity of the EF3-incubated RIF cells
plotted versus the O2 concentration of
drug incubation. The curve was calculated based on the fluorescence
intensity at each oxygen concentration, corrected for the ratio of
maximum (at <0.005% O2) binding at an actual
EF3 concentration of 175 µM and a 30-min
exposure (see Fig. 1
) to that from a 30 µM, 3-h
exposure. On the basis of this line, there was an 40-fold decrease
in fluorescence intensity as the oxygen concentration of drug
incubation increased from 0.005 to 10%. Most of this change occurred
between 0.1 and 1% O2. At oxygen concentrations
of 0.005, 0.1, 1, and 10%, Fig. 2
also depicts binding as found after
incubation with 14C-labeled EF3. At oxygen
concentrations from 0.005 to 1%, radioactive detection of EF3 closely
matched the indirect detection of EF3 with a fluorescent monoclonal
antibody. At 10% O2 the radioactive assay
detected 3-fold less EF3 binding than detection with antibody. This
occurs predominantly because of a lack of sensitivity in the
fluorescence assay.
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EF3 binding in PDT-treated tumors was studied during the 30-min PDT
illumination (75 mW/cm2, 135
J/cm2) or for the 30 min immediately after PDT
completion. Fig. 3
depicts representative flow cytometric histograms of a control tumor
(EF3 alone) and of a tumor incubated with EF3 during illumination.
Compared with the control, the PDT-treated tumor displays a marked
shift in the histogram toward higher fluorescence intensities,
indicating more cell-bound EF3 and lower oxygen tensions.
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as cumulative frequency histograms. Groups containing four to eight
mice each were treated with EF3 during PDT, treated with EF3 after PDT,
or treated as controls. During PDT, tumors demonstrated a significant
increase (P = 0.007) in tumor hypoxia, as
indicated by an increase in the averaged median fluorescence intensity
(±SE) from 17.1 ± 2.8 in control tumors to
48.9 ± 8.3 in tumors receiving EF3 during illumination.
However, this increase in hypoxia was found only during and not shortly
after the PDT illumination. Tumors labeled for hypoxia in the 30 min
after PDT demonstrated an average median fluorescence intensity of
18.3 ± 3.3, similar to the control median of
17.1 ± 2.8. Low levels of nonspecific antibody binding
were detected (median, 3.1 ± 0.3).
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. Table 1
lists the range of fluorescence intensities found at the
50th and 90th percentiles
in control and PDT-treated tumors, as well as the oxygen
concentration (in percentage and mm Hg) corresponding to these
fluorescence intensities. On the basis of median binding, tumors
exhibited oxygen tensions of 3.1–5.3 mm Hg under control conditions,
1.2–2.4 mm Hg during illumination, and 3.0–5.2 mm Hg after
illumination. When tumor oxygenation was calculated from the
90th percentile of binding, oxygen tensions were
0.76 mm Hg during illumination but 
0.91 and 1.2 mm Hg in control
tumors and tumor studied post-PDT, respectively.
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6
are images of frozen sections from a control tumor (Fig. 5
) and from a
tumor incubated with EF3 during PDT illumination (Fig. 6
). EF3 binding
is shown in red, where the intensity of the color indicates
of the severity of tumor hypoxia detected. Perfused tumor vasculature
is shown in green, whereas tumor regions demonstrating
neither Hoechst (perfusion) nor EF3 antibody fluorescence appear
black, to be distinguished from regions of the image not
containing tissue (shown in dark blue). A typical control
tumor (Fig. 5
) demonstrated limited, low levels of EF3 binding, with
most of this binding occurring in regions lacking Hoechst-stained
vessels, i.e., poorly perfused regions. PDT treatment of RIF
tumors created more extensive hypoxia, which varied in severity between
tumor and section examined. Fig. 6
depicts two sections from one
PDT-treated tumor. Both sections demonstrated more severe and
widespread hypoxia than that found in controls. However, a noticeably
greater area of the section was hypoxic in Fig. 6B
than in
Fig. 6A
.
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DISCUSSION |
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Tissue oxygen tensions, as determined by EF3 binding, agree well with published measurements of tissue pO2 in tumors of the same RIF strain. Tumor oxygen tensions have been measured with the Eppendorf pO2 Histograph in Photofrin-sensitized RIF tumors before PDT illumination and at specific times during the delivery of 75 mW/cm2; the median tumor pO2 was 3.9 mm Hg in controls, 0.7 mm Hg during the delivery of 0–5 J/cm2, and 2.8 mm Hg during the delivery of 20–50 J/cm2 (5) . On the basis of the median EF3 binding in this study, similar oxygen tensions of 3.1–5.3 mm Hg in controls and 1.2–2.4 mm Hg in tumors during illumination (75 mW/cm2, 135 J/cm2) were calculated. EF3 detection of hypoxia is an average over the illumination time, whereas the Eppendorf pO2 Histograph measures oxygenation only during needle tracking; therefore, EF3 binding reflects both the 0–5 J/cm2 and 20–50 J/cm2 measurements made with the pO2 Histograph. Furthermore, because EF3 binding is averaged over 30 min, instantaneous local variations in pO2 (such as may occur during PDT) may be even larger than measurements allow. In tumors exposed to EF3 after PDT, tumor oxygenation calculated from median binding recovered to 3.0–5.2 mm Hg. Although tumor oxygenation after PDT with these conditions has not been reported using the Eppendorf pO2 Histograph, oxygen measurements for other conditions do demonstrate a similar trend, i.e., recovery of oxygen to control levels or higher immediately following illumination (5) .
Using EF3-dependent fluorescence intensities, we calculated tumor oxygen tensions for binding at the 90th percentile. As expected, oxygen tensions at this binding level were substantially lower. It was found, however, that the majority of cells in control or posttreatment tumors exhibited oxygen tensions >1 mm Hg. In contrast, tumor oxygen tensions as low as 0.05 mm Hg were calculated based on 90th percentile EF3 binding during PDT illumination.
The use of hypoxia markers to study PDT-created changes in tumor
oxygenation offers several benefits. Because hypoxia marker binding
increases as tumor oxygenation decreases, this technique provides an
increasing signal with decreases in oxygen concentration. In the RIF
line, for the EF3 incubation conditions used, oxygen concentrations
from 0.005 to 1% O2 could be discriminated by
antibody binding and from 0.005 to 10% could be discriminated by
radioactive assay. This range of O2
concentrations may be most important for investigating the consequences
of oxygen depletion during PDT. Others have found that at oxygen
concentrations from 0.5–1%, cell inactivation by PDT is reduced to
half of its value under normoxia (24
, 25)
. Cells under
anoxic conditions (the equivalent to <0.005% for this study)
demonstrate no response to PDT. Thus, the identification of populations
of hypoxic tumor cells during PDT illumination could be valuable for
understanding tumor responses to a treatment protocol.
Another benefit of hypoxia markers is that these drugs enable the investigation of spatial distributions of oxygen within a tumor. The imaging of tumor hypoxia after PDT has been studied by other investigators using different hypoxia-labeling drugs. Immunohistochemical analysis of binding of the hypoxia marker 7-(4'-(2-nitroimidazol-l-yl)-butyl)-theophylline by van Geel et al. (14) demonstrated increased tumor hypoxia within 2 h after interstitial PDT of RIF tumors. Using Hoechst to label patent blood vessels, van Geel et al. (14) detected decreases in tumor perfusion after PDT and showed that areas of 7-(4'-(2-nitroimidazol-l-yl)-butyl)-theophylline binding did not overlap with the remaining perfused areas. Moore et al. (15) used the marker [123I]iodoazomycin arabinoside to monitor tumor hypoxia after PDT by noninvasive nuclear scintigraphy; a significantly higher relative retention of [123I]iodoazomycin arabinoside could be detected in tumors 24 h after PDT than in corresponding controls. In the present work, we demonstrate the ability to label tumor hypoxia and perfused blood vessels in control and PDT-treated tumors. The PDT-treated tumors clearly demonstrated more intense EF3 binding, i.e., more severe hypoxia, as well as binding over a larger tumor area. Future studies will evaluate these and associated images more closely in an attempt to quantify the findings.
A consideration in using hypoxia markers for study of oxygen tensions in PDT-treated tumors is the possible effect of PDT-created reductions in vascular perfusion on EF3 delivery to tumors. Significant reductions in vascular perfusion during or after PDT could limit EF3 availability to the tumor, resulting in lower levels of EF3 binding than expected. To assess vessel patency during or after PDT, markers of perfusion can be used in conjunction with EF3 to simultaneously study tumor perfusion and hypoxia (14) . In this study, qualitative comparison of Hoechst-stained blood vessels in control and PDT-treated tumors suggested that most tumors retained significant perfusion at the conclusion of treatment, indicating that although some changes in tumor perfusion may have occurred, widespread shutdown of tumor vasculature was not visible during PDT. Images of tumors exposed to EF3 after PDT (data not shown) reveal that although low levels of EF3 binding were found after PDT, this binding was well distributed throughout the tumor, indicating good perfusion of EF3 throughout the tumor. Furthermore, using the same treatment conditions of 5 mg/kg Photofrin, 135 J/cm2 at 75 mW/cm2, Fingar et al. (3) found no PDT-associated constriction or increased permeability in the vasculature of rat cremaster muscle up to 1 h after illumination. Therefore, the changes in EF3 binding found in this study are most likely associated with the depletion of oxygen by photochemical consumption, with less contribution from PDT-induced changes in vascular perfusion. Image analysis of Hoechst fluorescence will be used to quantify vascular perfusion in these tumors.
The use of hypoxia markers to study tumor oxygenation during PDT illumination required altering typical hypoxia marker protocols. Previously, 2-nitroimidazole exposure times in rodents were at least 2–3 h to allow adequate time for drug binding (20 , 26) ; in this study a 30-min EF3 exposure was chosen to correspond to a 30-min PDT illumination time. This short incubation time was further complicated by the fact that PDT can create changing oxygen concentrations over the course of illumination (1 , 5) . To confirm that tumor hypoxia during this 30-min incubation would be accurately represented by EF3 labeling, binding was studied as a function of time for exposures ranging from 1.5 to 60 min; drug binding was linear over this range. To confirm that EF3 binding, as detected indirectly through a fluorochrome-labeled antibody, corresponded to the actual EF3-cellular adducts formed, the fluorescence assay was compared to direct detection of EF3 using a radioactive assay. Good agreement between the two assays was found, although radioactivity permitted better detection of low EF3 binding at 10% O2. For the exposure conditions used, the fluorescence assay could detect a maximum oxygen concentration of 1% because of limitations in the sensitivity of antibody staining and flow cytometry.
An in vitro curve was used to determine oxygen concentrations corresponding to EF3 binding levels. In creating this curve, precision gas changes in valve-controlled airtight chambers and sustenance of cells in a minimum volume of medium helped to ensure that the plated cells were equilibrated to the intended oxygen concentration (22) . Hypoxia marker binding resulting from this technique is highly reproducible as shown previously (17) and demonstrated within this study. However, EF3 binding in plated cells at <0.005% O2 in these chambers does not perfectly correlate with EF3 binding in cell suspensions (in spinner flasks) at <0.005% O2 for the time course experiments. In particular, 30 min of 175 µM EF3 exposure in the time course experiments produced a median fluorescence intensity of 240, whereas exposure to 30 µM EF3 for 180 min in the chambers resulted in median EF3 binding of 497. The most likely explanation for this discrepancy is that more EF3 binding occurred during the chamber incubations because of binding during the 30-min gas exchanges at room temperature and the subsequent 15 min to bring the chambers to 37°C, which were not considered part of the 180-min incubation time (at 37°C). In addition, although EF3 binding is linear with time of incubation, binding is likely not as linear with drug dose; therefore, a 6-fold decrease in drug dose may have been slightly overcompensated for by a 6-fold increase in incubation time. Because times of EF3 incubation could be more finely controlled for exposures in cell suspension, as in the time course experiments, these experiments were used to estimate the maximum EF3 binding (at <0.005%) expected for the in vivo exposure conditions (30 min at 175 µM). Intermediate oxygen concentrations could not as accurately be studied in cell suspension, however, so relative changes in binding from exposure at different oxygen concentrations were studied using the chambers. Thus, the final curve of fluorescence intensity versus oxygen concentration incorporated data from both types of studies to produce the most accurate representation of absolute EF3 binding at different oxygen concentrations that is possible with our current methods.
In summary, this work demonstrates that EF3 binding can be used to detect and discriminate between tumor hypoxia developing during PDT illumination and that found before or after PDT. A method for calculating tissue oxygen tensions from EF3 binding levels is presented and shown to produce values in good agreement with published data.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Salary support for T. M. Busch was provided
by NIH Training Grant CA 09677. 
2 To whom requests for reprints should be
addressed, at Department of Radiation Oncology, University of
Pennsylvania, 195 John Morgan Building, 3620 Hamilton Walk,
Philadelphia, PA 19104-6072.
3 This author published previously under the name
Theresa M. Sitnik.
4 The abbreviations used are: PDT, photodynamic
therapy; EF3,
2-(2-nitroimidazol-1[H]-yl)-N-(3,3,3-trifluoropropyl)acetamide;
EF5,
2-(2-nitroimidazol-1[H]-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide;
RIF, radiation-induced fibrosarcoma; PF, paraformaldehyde.
5 Available at www.bio.umass.edu.
Received 11/ 4/99. Accepted 3/16/00.
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REFERENCES |
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B. W. Pogue, J. A. O'Hara, E. Demidenko, C. M. Wilmot, I. A. Goodwin, B. Chen, H. M. Swartz, and T. Hasan Photodynamic Therapy with Verteporfin in the Radiation-induced Fibrosarcoma-1 Tumor Causes Enhanced Radiation Sensitivity Cancer Res., March 1, 2003; 63(5): 1025 - 1033. [Abstract] [Full Text] [PDF]
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L. K. Folkes and P. Wardman Enhancing the Efficacy of Photodynamic Cancer Therapy by Radicals from Plant Auxin (Indole-3-Acetic Acid) Cancer Res., February 15, 2003; 63(4): 776 - 779. [Abstract] [Full Text] [PDF]
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T. M. Busch, E. P. Wileyto, M. J. Emanuele, F. Del Piero, L. Marconato, E. Glatstein, and C. J. Koch Photodynamic Therapy Creates Fluence Rate-dependent Gradients in the Intratumoral Spatial Distribution of Oxygen Cancer Res., December 15, 2002; 62(24): 7273 - 7279. [Abstract] [Full Text] [PDF]
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M. I. Koukourakis, A. Giatromanolaki, J. Skarlatos, L. Corti, S. Blandamura, M. Piazza, K. C. Gatter, and A. L. Harris Hypoxia Inducible Factor (HIF-1a and HIF-2a) Expression in Early Esophageal Cancer and Response to Photodynamic Therapy and Radiotherapy Cancer Res., March 1, 2001; 61(5): 1830 - 1832. [Abstract] [Full Text]
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) by antibody staining and flow
cytometry. 14C-EF3 binding was detected by measuring
radioactivity (
). To permit comparison of relative levels of EF3
binding as detected by radioactivity versus
fluorescence, the radioactive data were normalized to the EF3 binding
at 0.005% and then plotted such that relative binding at 0.005%,
i.e., 1, lined up with the fluorescence
intensity detected at 0.005%. All data points represent the
mean ± SE (bars) of two to three
separate experiments. The curve was drawn by correcting each
fluorescence intensity for the ratio of maximum binding possible at a
175 µM, 30-min exposure (from Fig. 1
) and a tumor treated with PDT (
). EF3 (52
mg/kg) incubation was for 30 min; tumors were then enzymatically
digested, stained with a monoclonal antibody, and read by flow
cytometry. In the PDT-treated (5 mg/kg Photofrin) tumor, EF3 incubation
was carried out during the illumination (75 mW/cm2, 135
J/cm2).
).
Nonspecific antibody binding (
) was measured in RIF tumors not
exposed to EF3 but stained with antibody. All EF3 exposures were for 30
min, followed by tumor excision, digestion, antibody staining, and flow
cytometry. In PDT-treated (5 mg/kg Photofrin, 75 mW/cm2,
135 J/cm2) tumors, EF3 was administered within 3 min before
illumination, with drug incubation during illumination, or within 3 min
after illumination, with drug incubation for the ensuing 30 min.
Control conditions included exposure to EF3 alone, EF3 plus
illumination (no Photofrin), or EF3 plus Photofrin (no illumination);
these controls all produced similar levels of EF3 binding and were
averaged for graphing. Data points represent the mean ± SE (bars); four to eight animals per group.
