Photod namic Thera y (PDT of solid tumors with I'd-bacteriophco horbidc
(WST095 Functional &aging !ly Blood Oxygen Level-Depcndent (RJLD) MKI
§Shimon Gross',' ,§Assaf Gilead' Avigdor Scherz', Michal Neeman' and 'Yoram Salornon'
The Departments of 'Biological Regulation and iPlant Sciences, The Weizmann Institute of Science, Rehovot 76100, Israel
Abstract
Photodynamic-therapy (PDT) relies on intravenous hotosensitizer
administration followed b local tumor illumination. tytotoxicity is
conferred by light-dependient, in-situ generation of reactive oxygen
species. As ,a consequence, PDT results in oxygen consum tion and
hemodynamic alteratlons. In order to evaluate treatment effiPcienc in
real-time, Gradient-echo MRI was applied to monitor changes in b&od
ox en levels and tissue hemodynamics. Intense, profound decrease
(2$50%) in MR signal intensity was observed in mouse melanoma
Pd-bacteriopheophorbide-PDT., Results were
tumors durin
independentfy validated by intravital microscopy and
immunohistopathology. It IS suggested that MRI may be applied for
guidance and efficiency assessment of PDT.
Introduction
Photodynamic therapy (PDT) relies on the destruction of tumors by insitu generation of cytotoxic reactive oxygen species (ROS) following
photosensitizer administration and tumor illumination. In previous
studies we reported that immediate illumination after i.v injection of
hacteriochlorophyll-serine to melanoma bearing mice, leads to tumor
oxygen depletion, capillary occlusion, hemorrhage and blood stasis,
tumor necrosis and erradication'.'. This novel anti-vascular treatment
modality was shown to induce high cure rates (-80%) in mice' and
rats3. Delivery of PDT to internal organs by optic fibers requires high
precision. While fiber insertion can be assisted by various techniques
(optical, X-ray or ultrasound), real-time visualization of tumor response
is presently not available.
Gradient echo (GE) MRI was previously shown to be a sensitive tool
for assessing hemodynamic changes in tumors4, and therefore was
applied, in this study, to evaluate tumor vascular response during and
after WST095 based PDT using M2R mouse melanoma as a model. We
suggest that such a technique can provide the means for real-time
functional guidance and efficiency assessment of the local therapeutic
photodynamic effect.
Materials and Methods
Animal and tumor model: M2R melanoma xenografts were
subcutanously implanted in male CDI-nude mice (4 weeks old, 30g
body weight). PDTMRI were performed when tumors reached 7-12
mm in diameter. Mice were anesthetized with 100 mg kg-' Ketamine
and 4 mg kg-' Xylasine (75% intraperitoneal and 25% subcutanonsly).
PDT of tumors: WST09 (by NEGMA-LERADS, France) administration
and tumor illumination were performed as described earlier6 inside the
magnet during MRI recordin$ Tumors were illuminated (763nm, 210J
cmz for 10 minutes, 0=lcm immediately after i.v administration of
WST09 ( 5 mg kg-I). using a 1W diode laser (CERAMOPTEC,
Germany)
Controls: Light control, illuminated tumors in mice administered with
vehicle. Dark control, WST09 ( 5 mg kg.') administered without
illumination.
MRI Studies: Gradient echo images were acquired on a horizontal 4.7-T
Brnker-Biospec spectrometer (Germany) using an actively RFdecoupled 1.5-cm surface coil and a whole body transmission coil.
Imaging parameters were: slice thickness of 0.8 mm, TR 200 ms, TE 10
ms, and 256 x256 pixels, in plane resolution of 136, 15 evolution
cycles, 2 averages each.
Analvsis of the MRI data: MRI data analysis was performed using
Scionimage software (Scion Corp. USA) and Orisis software6 (ver
4.0.9, University hospital, Geneve, Switzerland). MR signal intensity
was derived from each image either from region of interest inside the
tumor or in control tissue (muscle in opposite limb) and normalized to
the first image. Response maps were generated as a division of each
image by the average of the first 2 pre-PDT images.
Q Equally contributed to this work.
t To whom correspondence should be sent.
© Proc. Intl. Soc. Mag. Reson. Med. 10 (2002)
Intravital microscoov: Mice were anesthetized (as above) and
administered (i.v) with TRITC-dextran (500Kd, 3mg/animal, Sigma
corp.) as a blood-pool marker. Intravital fluorescence microscopy
(Axiotop-35 inverted fluorescent microscope, Zeiss-Germany, equipped
with a digital camera, DVC Corp. USA) was used to study
hemodynamics in real-time. PDT (conditions as above) was performed
on the central area of the mouse ear, on top of the microscope stage.
Fluorescence images were acquired every 2 minutes, before, during and
up to 15 minutes after illumination.
Results
In all PDT treated mice, profound decrease of the BOLD signal was
observed (2550%). This reduction in signal intensity was light and
WST09 dependent, confined to the illuminated tumor area. No
significant change in signal intensity was observed in the nonilluminated control tissue. Response maps representing signal
alterations before during and after PDT revealed the temporal and
spatial dynamics of the process. Upon onset of illumination, (during the
first 2 min) signal reduction was primarily localized at the light beam
impact zone. As illumination proceeded and for at least 40 minutes after
the light was switched off, signal reduction was observed mainly in the
tumor rim. WST09-PDT-induced hemodynamic changes were verified
by intravital fluorescence microscopy. As soon as 2 minutes after onset
of illumination, vascular occlusion, increased vascular dye leakage and
vasoconstriction were observed as previously reported6. No such
changes were observed in the controls. Tumor vascular photodamage
was independently validated by histopathology
and
immunohistochemistry of lipid peroxidation*. Tumor necrosis (24 h
post treatment) was verified by visual examination and
histopatholology.
Discussion
WST09-based PDT is a highly potent local anti-tumor treatment
modality. The acute antivascular effects of this treatment (reduced
blood oxygenation and hemodynamic alterations) were shown to
directly influence the GE-MR signal. Application of MRI during PDT
will enable better understanding of the role of vascular photodamage in
tumor eradication and cure. Moreover, from clinical perspective, this
method may assist the development of high precision MRI guided PDT
protocols. Such a technique may provide accurate treatment delivery.
Supported by STEBA BIOTECH N K The Netherlands.
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