Tumor Oxygen Dynamics During Photodynamic Therapy
Brian W. Pogue1*, Julia A. O'Hara2, Carmen J. Wilmot2 and Harold Swartz2
1Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire,
03755.
2Department of Radiology, Dartmouth Medical School, Hanover, New Hampshire
03755.
INTRODUCTION: Photodynamic therapy (PDT) is a treatment for diseased
tissues which is currently
approved in the US for clinical treatment of late stage esophageal cancer
treatment, age-related
macular degeneration, some skin cancer tumors and is undergoing clinical
trials for numerous
other cancers and non-cancerous conditions. Most photosensitizers have
similar physical
chemistry, producing singlet oxygen in high yield when excited by light.
However, the
mechanisms behind tissue destruction in PDT are complex and unlikely to be
described by any
one process. In contrast to the similar photochemical processes of different
photosensitizers, the
biological action of these photosensitizers is thought to vary considerably.
The pathways of
biological damage are thought to originate from massive singlet oxygen
insult, which causes the
following possible reactions (i) acute cellular necrosis, (ii) acute
vascular damage, (iii) apoptosis,
and (iv) later onset of inflammatory reactions. One of the features of
photosensitizers that can
alter the mode of tissue destruction is that the compartmentalization of the
molecules can change
over time. This can result in different sites of photodamage, depending upon
the time between
injection and optical irradiation treatment. The oxygen dynamics during PDT
were examined
here to differentiate the response of vascular-targeting therapy from
non-vascular targeting
therapy.
METHODS: In this study, the vascular and tissue oxygen changes
induced by photodynamic therapy
in the RIF-1 tumor were examined in vivo using L-band electron paramagnetic
resonance (EPR)
oximetry with implanted lithium phthalocyanine. Two photosensitizers,
including verteporfin
(benzoporphyrin derivative in a lipid-based formulation) and aminolevulinic
acid-induced
protoporphyrin IX (ALA-PPIX), were investigated with optical irradiation
sufficient to induce
sub-curative damage in the tumor tissue, and the transient changes in pO2
and vascular perfusion
were examined. The cellular response to treatment was also examined in vitro
using X-band
spectroscopy with nitroxides to measure changes in the the cellular oxygen
consumption rate in
response to therapy. For vascular sensitization, verteporfin was injected at
1 mg/kg into mice
bearing subcutaneous RIF-1 tumors 15 minutes prior to irradiation. Tumor pO2
was monitored
with the L-band throughout treatment and at 1, 2, 4, 8, 24 and 48 hours post
treatment. The same
measurements were carried out in animals tumors treated with non-vascular
targeting therapy,
which was based on waiting for 3 hours between injection and irradiation,
using both verteporfin
and ALA-PPIX photosensitizers.
Cellular respiration was monitored in vitro using RIF-1 cells grown in
culture and
centrifuged down to a concentration of 1x107 cells per ml, in media with 10%
dextran. Nitroxide
was added to the suspension and oxygen concentration was monitored over a 20
minute period
and the consumption rate was measured as the slope of oxygen concentration
versus time.
Samples of cells were taken from the same suspension and treated with
verteporfin for 3 hours
and irradiated with an LD90 light dose and then placed in the EPR resonator
for measurement of
oxygen consumption.
RESULTS: In vivo Tumor Oxygen
A large increase in tissue oxygenation (from 3 mm Hg up to 9.5 mm Hg) was
observed when
treated with ALA-PPIX based photodynamic therapy, which lasted only during
the treatment and
a small residual increase that returned back to baseline levels by 48 hours
after treatment. With
verteporfin-based photodynamic therapy, one group of animals was irradiated
15 minutes after
injection and had a small decrease in oxygenation relative to
pre-irradiation levels. The second
group was irradiated at 3 hours after injection and had a large increase in
the average pO2, (from
3 mm Hg to 15 mm Hg) by the end of the treatment. These observations
indicate that
photodynamic therapy significantly increases tissue pO2 under certain
treatment conditions, with
the potential cause being either increased local blood flow or decreased
local oxygen metabolic
consumption due to cellular damage.
In Vitro Oxygen Consumption Rate
The oxygen consumption rate in vitro was reduced by a factor of 4 in cells
treated with PDT
using verteporfin. NADH fluorescence was monitored with confocal microscopy
and this
indicated a reduction in this mitochondrial metabolite.
SUMMARY: These observations indicate that tumor cellular oxygen
consumption rate is significantly reduced
immediately after therapy. This observation could be partially responsible
for the increase in
tumor pO2 during non-vascular targeting application of PDT, as observed in
this study. The
implications of these observations are important because they suggest that
PDT can be used to
increase tumor oxygenation under certain circumstances, and may be a very
useful
radiosensitizer. In addition, increases in tumor oxygenation into regions of
chronic hypoxia are
possible and may lead to increased sensitization of those areas with
low-dose rate application of
PDT. Further investigation of this effect and the role of vascular stasis
and blood flow shunting
mechanisms are needed to fully explain the phenomenon observed in vivo.