Photodynamic therapy of C6-implanted glioma cells in the rat brain employing second-generation photosensitizer talaporfin sodium

Photodiagnosis and Photodynamic Therapy (2008) 5, 198—209

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Photodynamic therapy of C6-implanted glioma cells in the rat brain employing second-generation photosensitizer talaporfin sodium Hiroaki Namatame MD a, Jiro Akimoto MD a,∗, Hiroyuki Matsumura MD a, Jo Haraoka MD a, Katsuo Aizawa PhD b a

Department of Neurosurgery, Tokyo Medical University, Japan Department of Physiology, Tokyo Medical University, Japan Available online 22 October 2008

b

KEYWORDS Photodynamic therapy; Talaporfin sodium; Malignant glioma; Transplanted rat glioma model; C6 glioma cell line

Summary Object: The usefulness of photodynamic therapy (PDT) as a local therapy for malignant glioma was evaluated by investigating histological changes in a rat C6 glioma model treated with a combination of talaporfin sodium, a water-soluble photosensitizer derived from chlorophyll and exposure to a diode laser. Methods: Glioma cells (C6) at the confluence stage were transplanted stereotactically into the right frontal lobe of SD rats. Five days later, the rats underwent right frontal craniotomy and intravenous administration of talaporfin sodium. One hour after talaporfin sodium administration, each rat was irradiated by a 664 nm diode laser beam. The brain was removed 1, 3 or 6 h after laser irradiation for histological examination of tumor-affected brain tissue and surrounding normal brain tissue. Results: In addition to the tumor mass, tumor cells invading surrounding edematous brain tissue were seen in untreated rats, ranging from the brain surface to a depth of 2 mm. One hour after PDT, coagulation necrosis as well as disappearance of indication of cell viability such as disappearance of tumor cell processes and foamy changes of cytoplasm were noted in the tumor tissue at a depth of 0.5 mm, accompanied by reduction of cytoplasmic glial fibrillary acidic protein (GFAP) expression and appearance of granular M30 cytodeath positivity. Three hours later, the cytoplasm of the residual tumor cells showed disappearance of GFAP expression and increased expression of M30 cytodeath. Six hours later, the foamy cytoplasm of swollen tumor cells demonstrated strong positivity for M30 cytodeath. Conclusion: PDT using talaporfin sodium induced coagulation necrosis and apoptosis in rats with C6 glioma. © 2008 Elsevier B.V. All rights reserved.

∗ Corresponding author at: Department of Neurosurgery, Tokyo Medical University, 6-7-1 Nishishinjuku, Shinjuku-ku, 160-0023 Tokyo, Japan. Tel.: +81 3 3342 6111x5773; fax: +81 3 3340 4285. E-mail address: [email protected] (J. Akimoto).

1572-1000/$ — see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.pdpdt.2008.08.001

Photodynamic therapy of C6-implanted glioma cells in the rat brain

Introduction It is essential to improve surgical respectability of malignant glioma [1—4]. Neuro-oncologists have striven to improve resectability using all possible modalities, including functional and anatomical evaluation, intraoperative navigation, electrophysiological monitoring, intraoperative MRI. However, this tumor is likely to recur from the operated site within several months, and in cases of glioblastoma it is difficult to achieve a mean survival period longer than 2 years [1,2,5]. Unlike tumors of other organs, since malignant glioma is less likely to metastasize, satisfactory local control is essential [3—5]. The quality of initial surgery is, therefore a key factor determining the success of treatment. Major factors rendering curative resection of malignant glioma difficult are peritumoral cerebral function and tumor invasiveness [1—5]. To resolve the former problem, surgeons try to resect the affected area as extensively as possible, making full use of brain monitoring and taking care to avoid functional morbidities. Despite such attempts, complete tumor resection may hardly be achieved [2,3]. The latter factor presents the greatest problem. Tumor cells frequently infiltrate 2—3 cm from the main tumor mass [4]. If tumor cells in this area would be controlled, the risk of tumor recurrence might be reduced. Early in the 1980s, attempts began to be made to treat malignant glioma by inducing selective apoptosis of tumor cells through preoperative administration of a photosensitizer likely to be specifically incorporated into tumor cells, and intraoperative application of a light which can excite the photosensitizer and penetrate tissue [6—8]. This therapy is called photodynamic therapy (PDT) because it depends on the cytotoxic effects of singlet oxygen formed through photochemical reactions of the photosensitizer [9,10]. PDT has been proposed to serve as an approximately ideal operative procedure for malignant glioma if it can selectively control residual tumor tissue in the functional brain area or the tumor cells invading the normal brain tissue, while preserving the morphological and functional neuronal structures [11,12]. In the past, many neuro-oncologists were attracted by the theory of PDT and tried to apply PDT in the preclinical or clinical stage [6—8,11—19]. To date, however, the therapeutic value of PDT, although reported for cancer of other organs, has rarely been demonstrated for malignant glioma. Considering the known mechanism of PDT efficacy, a key point in elevating its therapeutic value is how to achieve formation of singlet oxygen in amounts large enough to cause specific damage to malignant glioma cells [12,13,15,17,18,20—23]. However, few reports published to date showed that the therapeutic efficacy of photosensitizers such as porfimer sodium is enough to offset its disadvantages (the necessity of care for phototoxicity such as skin photosensitivity) [7,8,14,17,19,24—27]. Furthermore, equipments needed are expensive. At present, PDT is not widely accepted for the treatment of malignant glioma. Ideal photosensitizers are those which have high tumor cell selectivity, cause minimum phototoxicity and are likely to induce formation of singlet oxygen when stimulated by appropriate exciting light. Talaporfin sodium is a second-generation photosensitizer developed in 1987 by Aizawa et al. [28]. This substance,

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derived from plant chlorophyll, has a chlorine ring frame characterized by attachment of one aspartate molecule via a peptide bond and by replacement of all four carboxylic acids of the side chain with sodium salt [28,29] (Fig. 1A). It is a dark green powder, with a molecular weight of 799.7. In the living body, talaporfin sodium binds to albumin at a molar ratio of 1:1 [28,30]. It does not pass the blood brain barrier. Regarding the wavelengths for its absorption, it is known that this compound has a Soret band at 405 nm and that it has a maximal Q band at 664 nm (a wavelength longer than that for porfimer sodium) [28—30] (Fig. 1B). Because of these features related to absorption, talaporfin sodium penetrates more deeply into tissue than conventional photosensitizers. Furthermore, talaporfin sodium is eliminated rapidly from normal tissue and is less likely to cause adverse reactions such as photosensitivity [28]. In Japan, the safety and efficacy of this compound in the treatment of early lung cancer have been demonstrated in clinical trials. In June 2004, it was adopted for coverage by the Japan national health insurance for lung cancer. We previously reported the tendency of this compound to selectively accumulate in glioma tissue disrupting the blood brain barrier [29]. In addition, numerous reports have been published concerning the specificity of the distribution of this drug in tumor cells of various cancers [29,31—36]. Based on these findings, we considered that talaporfin sodium is an optimal photosensitizer for PDT for malignant glioma, and in this study sought to evaluate the feasibility of PDT for malignant glioma primarily based on pathological studies of a rat glioma model.

Materials and methods Talaporfin sodium (mono-L-aspartyl chlorine e6, NPe6) Talaporfin sodium is a second-generation photosensitizer. It has a chlorine ring structure in which one of the double structures on ring D of a tetrapyrol ring has been cleaved [28]. At the carbon 15-position, aspartate is attached to one side chain by means of an amide bond. It is a hydrophilic photosensitizer with a molecular weight of 799.7 and can be characterized by rapid elimination from normal tissue (Fig. 1A). In phosphate buffer (pH 7.4), talaporfin sodium showed absorption peaks in the Soret zone (398 nm) and the Q zone (502, 530, 620 and 654 nm) [28—30]. The peak of its longest absorption spectrum is located at 654 nm, in the over-600 nm range where absorption by hemoglobin is unlikely. If talaporfin sodium is excited at its absorption wavelength, competition with hemoglobin which is absorbed at 576 nm is minimal, allowing observation of a fluorescence spectrum in vivo with a peak at 662 nm [28,29]. When it undergoes structural changes due to binding to or replacement with albumin, lipoprotein, etc., the tetrapyrol ring absorption zone after incorporation into tumor shows a bathochromic shift of about 10 nm longer wavelength [28—30]. If excited at the absorption wavelength 664 nm during this change, a specific fluorescence image with a peak at 672 nm can be obtained (Fig. 1B).

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Figure 1 (A) Chemical structures of talaporfin sodium (mono-L-aspartyl chlorine e6, NPe6) N-[[(2S,3S)-18-carboxy-2-(2carboxyethyl)-13-ethyl-2,3-dibydro-3,7,12,17-tetramethyl-8-vinylporphyrin-20-yl]acetyl]-L-asparatic acid. (B) Absorption spectrum of talaporfin sodium and change in absorption wavelength following conjugation with albumin (solid line; talaporfin sodium and phosphate buffer solution, dotted line; talaporfin sodium conjugated with albumin) talaporfin sodium has absorption peaks in the Soret band (398 nm) and Q bands (502, 530, 620, and 654 nm) in pH 7.4 phosphate buffer solution (PBS). When it conjugates with albumin, its absorption band wavelength becomes approximately 10 nm longer (bathochromic shift).

Rat glioma model C6 glioma cells during logarithmic proliferation in RPMI 1640 medium supplemented with 10% fetal calf serum were used. Cells were treated with trypsin immediately before transplantation, to yield single cell suspensions (5 × 105 /5 ␮l). Under intraperitoneal Nembutal anesthesia, 10-week-old male SD rats (body weight 330—370 g, N = 5) were allowed to breathe spontaneously, with the body temperature kept at 37 ◦ C. The head of each rat was immobilized in a stereotactic brain surgery device (SR-5R, Narishige Co., Ltd., Tokyo, Japan). A small hole was created with a drill on the right coronal suture, 2 mm from the midline of the skull, followed by a small incision of the dura mater. Then, using a 27-gauge Hamilton syringe, the brain was punctured slowly with a needle to a depth of 1.5 mm, with care taken to minimize injury of the brain parenchyma. The above-mentioned C6 glioma cells (5 ␮l) were then transplanted to create the intracerebral C6 tumor model.

performed to the craniotomized area. The PDT excitation was provided by a 664 nm diode laser generator (Matsushita Industrial Equipment Co., Ltd., Osaka, Japan) composed of diode laser elements (made of AlGaInP), a temperature adjuster (cooling device) to control the oscillating wavelength of the diode laser elements, a unit to control these devices, an energy input unit and an indicator. The central wavelength for diode laser oscillation was set at 664 nm by means of temperature control. To narrow the half-band width of the spectrum at wavelength, a visible light band pass filter centered on a wavelength 664 nm (Koshin Kogyo Co., Ltd., Osaka, Japan) was attached to the laser outlet side. Using a fiber with a Selfoc lens attached its tip, exciting light was applied to an area with a diameter of 10 mm with uniform power (100 mW/cm2 , 10 J/cm2 ), as shown in Fig. 2. The control group (sham operation group) received

Administration of talaporfin sodium Five days after creation of the C6 glioma model, the i.p. Nembutal-anesthetized animals underwent craniotomy of the right half of the skull, with a high-speed drill, during spontaneous respiration, with the body temperature kept at 37 ◦ C. The dura mater was incised carefully, avoiding injury of the venous sinus. In this way, the right cerebral hemisphere was exposed as widely as possible to check for tumors from brain surface. Then, talaporfin sodium (5.0 mg/kg: optimal dose of administration for uptake and retention in glioma cells [29]) dissolved in 1 ml of physiological saline was injected slowly via the tail vein.

PDT protocol For 1 h after administration of talaporfin sodium, the stability of vital signs in each rat was monitored, then PDT was

Figure 2 Photodynamic therapy for rat C6 glioma model diode laser (100 mW/cm2 , 10 J/cm2 ) irradiated to the craniotomoized rat brain by full-circumferential irradiation probe (arrow: probe for irradiation of diode laser).

Photodynamic therapy of C6-implanted glioma cells in the rat brain irradiation with the same laser, without administration of talaporfin sodium (N = 5).

Histological studies After PDT, the rats were managed while confirming the stability of vital signs under craniotomy, using additional intraperitoneal doses of Nembutal as needed. The time course of histological changes of the brain after PDT was followed by carefully removing the brain from rats sacrificed 1, 3 and 6 h after PDT (N = 5 each time). Brains fixed in 10% formaldehyde were halved coronally along the tumor injection trace and embedded in paraffin before coronal sections were cut and stained with hematoxylene and eosin. Adjacent sections were stained with Luxol-fast-blue (LFB) and immunostained with a monoclonal antibody against the glial fibrillary acidic protein (GFAP: dilution 1:400, Dako Cytomation, Glostrup, Denmark) and M30 cytodeath (dilution 1:100, Boehringer Mannheim GmbH, Mannheim, Germany). For immunohistochemical examination, tissue sections (6 ␮m) were deparaffinized, followed by removal of endogenous peroxidase. Then, they were exposed to microwave within a citrate buffer (pH 6.0) for 15 min to retrieve the antigen. At room temperature, the sections were exposed to anti-GFAP antibody and M30 cytodeath for 24 h. A two-step immunohistochemical staining technique (DAKO EnVision System, HRP-DAB, Dako Cytomation) was used for visualization.

Results Rat glioma model In the rat glioma model, brain tumors were formed in 100% of animals, ranging from the brain surface to a depth of 2 mm on average. The tumors generally had a semi-circular form. The main bulk of the tumor was composed of a dense distribution of tumor cells with numerous mitoses, oval nuclei and thin processes as well as micro-necrosis with pseudopalisading. In the border between the tumor and the brain parenchyma, neovascularization and tumor cells invading the brain, with some adhering to these newly developed vessels, were noted. The cytoplasm of the tumor cells was GFAP-positive but M30-negative. The area of the corpus callosum near the site of tumor transplantation was slightly edematous, but the fibrous structure of the myelin sheath was preserved (Fig. 3A—F). In the sham operation group where only laser was applied, no prominent histological change was noted in tumor tissue but slight edematous changes were noted in the surrounding brain tissue.

One hour after PDT The area from the brain surface to a depth of 1.5 mm within the tumor tissue consisted almost completely of eosinophilic amorphous necrosis-like structures. This area was partially interposed with cells with intensely stained nuclei suggesting tumor cells. The deepest layer of the tumor, where the tumor invaded the surrounding brain, also showed groups of such cells. These tumor cells were differed markedly from the morphological features of tumor cells seen in the

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sham operation group. They were round cells with condensed chromatin, displaced nuclei, foamy cytoplasm and lacked cell processes. Neither mitosis nor fragmentation of nuclei was visible in these cells. The cerebral cortex around the tumor showed edematous changes, resembling those seen in the sham operation group, but it showed no obvious morphological change of neuronal structures. In terms of morphological features, the tumor cells invading this area in the PDT group did not differ markedly from the control group. Positivity for GFAP was observed in the marginal area of the tumor cell cytoplasm in the PDT group, differing from the diffuse expression in the control group. M30 cytodeath expression was marked in the marginal area of the cytoplasm, showing a granular distribution. Staining of the area of the corpus callosum for LFB revealed no evident reduction of the myelin sheath (Fig. 4A—F).

Three hours after PDT The necrosis-like structure noted in the area from the brain surface to a depth of 1.5 mm became a complete tissue defect. Groups of tumor cells were noted only at a depth of about 0.5 mm where the tumor cells invaded the surrounding normal brain. The cells in this area were morphologically similar to the tumor cells noted 1 h after PDT. They had a round form, condensed chromatin and displaced nuclei. Pyknotic change of neurons and edematous change in the surrounding brain were more marked than those seen 1 h after PDT. Most tumor cells were negative for GFAP, but relatively intensely stained cell groups were noted in part of the marginal area of cytoplasm. Granular expression of M30 cytodeath in the marginal area of the cytoplasm, similar to that seen 1 h after PDT, was noted, and the responses were slightly more intense. LFB staining revealed the callosal fibrous structure of myelin sheath to be preserved, although there were evident edematous changes (Fig. 5A—F).

Six hours after PDT The deepest part of tumor tissue also assumed an eosinophilic, amorphous structure, containing only a very small number of cell groups with evident features of tumor cells. The tumor cells continued to have round, foamy cytoplasm with slightly condensed chromatin and dislocated nuclei. The pyknotic change of neurons in the surrounding brain tissue remained, but this change and edematous change were less marked than those observed 3 h after PDT. Expression of GFAP was generally absent in the small number of tumor cells remaining at that time. GFAP-positive structures were noted in a sporadic manner in the extracellular space. Intense M30 cytodeath expression was noted around the cytoplasm of tumor cells. M30 cytodeath was positive also in the amorphous area of the tumor mass. In addition, intense M30 cytodeath expression was also noted in the cytoplasm of tumor cells which had invaded the surrounding brain tissue. LFB staining revealed that the callosal fibers of the myelin sheath were preserved well and that the edematous changes in this area were milder than at 3 h after PDT (Fig. 6A—F).

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Figure 3 Photomicrographs of brain sections obtained in rats implanted with C6 glioma cells. (A) The semi-circular shaped tumor was formed in the range from the brain surface to a depth of 2 mm. The main part of the tumor was composed of high cellularity of tumor cells with focal necrotic change. The border between the tumor and adjacent brain shows infiltration of the tumor cells (hematoxylene and eosin, original magnification 20×). (B) The tumor bulk consisted of dense distribution of highly proliferative glioma cells with mitosis (hematoxylene and eosin, original magnification 200×). (C) The cytoplasm of the tumor cells presented in the tumor mass was strongly positive for GFAP (original magnification 100×). (D) The tumor cells did not express M30 cytodeath (original magnification 100×). (E) In the area of the brain—tumor interface, the tumor cells infiltrated to the brain parenchyma with neovascularization (hematoxylene and eosin, original magnification 100×, asterisk indicates the brain—tumor interface). (F) In adjacent cerebral cortex, neuronal structures was well preserved and slight proliferation of glial cells was noted (hematoxylene and eosin, original magnification 100×, arrow indicates the infiltrating glioma cell).

Photodynamic therapy of C6-implanted glioma cells in the rat brain

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Figure 4 Photomicrographs of representative brain sections obtained in C6 glioma rats, 1 h after treatment with talaporfin sodium and diode laser. (A) The area from the brain surface to a depth of 1.5 mm was composed of semi-circular amorphous eosinophilic tissues. In the deeper layer, the apoptotic tumor cells remained occupying a semi-circular area (hematoxylene and eosin, original magnification 20×, asterisk indicates the area of coagulation necrosis). (B) The remained tumor cells demonstrated the condensed and displaced nuclei, foamy cytoplasm and loss of cell processes. The cells were clearly apart from the control tumor cells (hematoxylene and eosin, original magnification 200×). (C) The expression of GFAP was remarkably reduced and located only in the marginal area of the cytoplasm of tumor cells (original magnification 200×). (D) The granular expression of M30 was noted in the cytoplasm of tumor cells (original magnification 200×). (E) The adjacent cerebral cortex demonstrated slight edematous change of the matrix, but the neuronal structures were well preserved (hematoxylene and eosin, original magnification 100×). (F) In the area of the corpus callosum, the myelin sheath was well preserved (Luxol-fast blue stain, original magnification 100×).

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Figure 5 Photomicrographs of representative brain sections obtained in C6 glioma rats, 3 h after PDT. (A) The tumor mass consisted of round tumor cells, which contained dense, displaced nucleus and foamy cytoplasm. Neither mitosis nor fragmentation of nucleus was noted in these tumor cells (hematoxylene and eosin, original magnification 200×). (B) The expression of GFAP was prominently reduced, and few cells demonstrated intense expression in the cytoplasm (original magnification ×200). (C) Remaining tumor cells intensely expressed of M30 cytodeath in cytoplasm (original magnification 200×). (D) The peritumoral cerebral cortex demonstrated mild edematous matrix and pyknotic changes of neuronal structures (original magnification 100×, arrow indicated morphological change of cortical neuron). (E) In the peritumoral cerebral cortex, the expression of GFAP was noted in the reactive gliosis (original magnification 100×). (F) In the area of the corpus callosum, the edematous matrix was noted but the myelin sheath was well preserved (Luxol-fast-blue stain, original magnification 100×).

Photodynamic therapy of C6-implanted glioma cells in the rat brain

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Figure 6 Photomicrographs of representative brain sections obtained in C6 glioma rats, 6 h after PDT. (A) Only small number of tumor cells were floating on the eosinophilic amorphous material, and they contained condensed, displaced nucleus and foamy cytoplasm (hematoxylene and eosin, original magnification 200×). (B) Almost all remaining tumor cells had reduced the expression of GFAP (original magnification 200×). (C) Strong expression of M30 cytodeath was demonstrated in the cytoplasm of tumor cells and amorphous matrix (original magnification 200×). (D) The edematous change of matrix and pyknotic change of neuronal structures were improved in the peritumoral cerebral cortex (original magnification 100×). (E) Granular expression of M30 cytodeath was also demonstrated in the cytoplasm of infiltrative tumor cells (original magnification 200×, arrow indicates cell damage of infiltrating glioma cell). (F) The area of the corpus callosum, the matrix demonstrated improvement of the edematous change of the matrix, and good preservation of the myelin sheath (original magnification 100×).

Normal morphology Area of corpus callosum

Normal morphology

Moderate edematous change with pyknotic change of neuronal structures Edematous matrix but preservation of myelin sheath Slight edematous change Slight edematous change

GFAP (++), M30 cytodeath (−) Infiltrative tumor cells with neovascularization Invading front

Peritumoral brain tissue

GFAP (+/−), M30 cytodeath (+) Infiltrative tumor cells with morphological change

Residual tumor cell nest with morphological change

Coagulation necrosis and morphological change of residual tumor cells GFAP (+), M30 cytodeath (+) Infiltrative tumor cells with morphological change Dense tumor cell proliferation with micronecrosis Tumor tissue Bulk

5 5 5 5 N

3 h after PDT 1 h after PDT Sham operation

We set out to establish the feasibility of PDT as a treatment for malignant glioma. According to our literature search, no previous reports deal with evaluation of talaporfin sodium as a means of PDT for malignant glioma. PDT efficacy depends on a number of factors, including oxygenation status, total light dose (fluence), the rate of light delivery (fluence rate), and photosensitizer concentration and localization [10,17,18,20,31,32,37,38]. Using our rat glioma model, we previously chronologically analyzed the intensity of fluorescence generated from tissue after administration of a photosensitizer and reported that the fluorescence intensity remained constant during the period from 45 min to 4 h after the administration via the tail vein [29]. Following this result, we planned to apply PDT 1 h after a dose of photosensitizer. Exciting laser was applied in the following way. Using a full-circumferential irradiation probe, exciting laser (664 nm) with a fluence rate of 100 mW/cm2 was applied to a 10 mm diameter circular field for 100 s (10 J/cm2 ) [9,31,34,36]. The factor having the greatest influence on PDT efficacy is known to be the laser fluence rate [10,13,19—21,31]. The fluence rate 100 mW/cm2 , employed in the present study, is equivalent to 10 times the fluence rate (10 mW/cm2 ) used for observation of fluorescence in rats with transplanted brain tumor we reported previously [29]. In previous clinical trials of PDT in patients with early lung cancer, the fluence rate was set at 150 mW/cm2 (100 J/cm2 ), but we adopted

Chronological histopathological changes of rat brain implanted C6 glioma cells after photodynamic therapy.

Discussion

Table 1

C6 glioma transplantation occurred at a depth of 2 mm from the brain surface. One hour after PDT, the tissue from the brain surface to a depth of 1.5 mm had changed into necrotic tissue almost completely, causing a subsequent tissue defect in this area. In the remaining deeper part (1.5—2.0 mm from the surface) of the tumor, tumor tissue remained and accompanied by invasion of surrounding brain tissue. However, at 1 h, the tumor cells in this area had already lost their original morphology and showed macrophage-like morphological features, such as lacking cell processes, with condensation of chromatin and displacement of nuclei and presence of foamy tissue within the cytoplasm. The tumor cell count decreased markedly over time after PDT, and only a very small number of tumor cell groups were noted 6 h after PDT. GFAP was strongly positive in the cytoplasm and process of tumor cells and M30 cytodeath was negative before PDT, but soon after PDT, the expression of GFAP decreased. As a result of this change, combined with morphological changes of tumor cells, the GFAP-positive area shifted to the edge of the cytoplasm, and expression decreased over time after PDT. M30 cytodeath expression was noted at the edge of the cytoplasm, resembling the expression pattern of GFAP observed soon after PDT, but its expression increased over time after PDT. In normal brain tissue, no marked change was visible soon after PDT, but pyknotic change of neurons around the brain tissue was observed 3 h after PDT, accompanied by edematous change of surrounding cerebral cortex and the area of corpus callosum. These changes had ameliorated 6 h after PDT (Table 1).

6 h after PDT

Summarized histopathological findings

Slight edematous change with preservation of neuronal structures Slight edematous change

H. Namatame et al. Small number of residual tumor cells with morphological change GFAP (−), M30 cytodeath (++) Infiltrative tumor cells with morphological change M30 cytodeath (+)

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Photodynamic therapy of C6-implanted glioma cells in the rat brain a fluence rate at 100 mW/cm2 because it was rat model. Needless to say, minimizing the effects on normal brain tissue is the most important consideration when applying PDT [12,37,39,40—42]. Yoshida et al. [41,42] applied an argonpumped dye laser beam (632 nm) to the brains of normal rats treated with photofrin at a fluence rate of 100 mW/cm2 (35 J/cm2 ) and reported the cell damage observed (including damage of vascular endothelium) in detail. Madsen et al. [22] applied a 635 nm diode laser beam (9—54 J/cm2 ) to rat normal brains after treatment with 5-ALA. They reported no morbidity at a fluence rate of 18 J/cm2 , but 50% mortality at 54 J/cm [20,22]. They additionally reported that necrosis of neurons and edema to a depth of 3 mm were also noted in the brains of the surviving rats treated with steroids [22]. Ligle and Wilson [14] applied 514 nm argon lasers (35 mW/cm2 , 50 J/cm2 ) to normal rabbit brains after treatment with one of four photosensitizers (5-ALA, photofrin, etc.) and evaluated apoptosis by TUNEL assay. They reported that necrosis in limited areas and background level apoptosis were the only abnormalities observed [15]. The diode laser used in the present study had a wavelength of 664 nm, longer than the wavelength of photosensitizers used in most reported studies, and this laser was expected to have a high potential of reaching deep layers of tissue. For these reasons, the total light dose (fluence) was set low (10 J/cm2 ) in the present study. As a result, no marked cell damage or edema was noted in the sham operation group. Considering the ability of talaporfin sodium to selectively accumulate in tumor cells [29,38,45], this photosensitizer is unlikely to affect normal brain. However, it is essential to adequately investigate the safety threshold for irradiation with diode lasers from now on. Photosensitizers which absorb light energy enter a higher energy state called the ‘‘excited singlet state’’ and then soon return to the basal state. They return to the basal state either directly or via an intermediate state (excited triplet state). In the former case, the energy gradient is converted into fluorescent energy, thus allowing PDD. In the latter case, oxygen radicals or free radicals are formed, allowing, for example PDT, which utilizes type 2 photochemical reaction, involving singlet oxygen [10,23,31,34,36]. PDT exerts its efficacy through two mechanisms: (1) direct effect based on the cytotoxic effect of singlet oxygen on tumor cells [9,10,15,34,36,43—47], and (2) indirect effect based on the induction of tumor necrosis through shut down of tumor vessels. [20,33,47]. For effective PDT, the photosensitizer must to be present in the target tissue of PDT. It is essential that the distribution of the photosensitizer is confined to tumor cells and endothelial cells of tumor vessels which have proliferative potentials [10,15,22,23,32,38,42,48]. Several reports on experiments of PDT using various models of transplanted cancer cells demonstrated that a major component of PDT effect is coagulation necrosis caused by vascular shutdown [33,48]. Yamamoto et al. [48] applied 664 nm diode lasers (100 mW/cm2 ) to the peritoneum of normal BALB/c mice immediately after a dose of talaporfin sodium. They reported that endothelial cell edema and chromatin condensation occurred 20 s after laser irradiation, and that fibrin thrombus formation was caused by activation of factor XIII [48]. In addition, they reported in detail the course of vascular obstruction occurring in the irradiated area within 80 s after irradiation [48]. Although damage of normal capil-

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laries due to PDT was observed in their study, Yamamoto et al. [48] added that adjustment of the interval between talaporfin sodium treatment and start of PDT changes the tissue distribution of the drug and with a clinically acceptable difference in vulnerability to PDT between tumors and normal vascular endothelial cells. In the present study, we also noted vascular coagulation necrosis in the upper 1.5 mm layer of the tumor bulk, which extended from the brain surface to a depth of 2 mm. No such vascular damage was noted in the deeper tumor tissue or in the normal brain tissue at the tumor-invading front. Because the severity of such coagulation necrosis depends on the conditions for PDT mentioned above, it is essential that the conditions for irradiation with the diode laser are determined, taking into account not only the influence on normal brain tissue but also that on normal brain vessels, as mentioned above, when attempting PDT for the most extensive invasion of malignant glioma in clinical cases. The mechanism for direct damage by singlet oxygen during PDT is determined by the distribution of the photosensitizer in the cells. Photosensitizers such as photofrin, 5-ALA and phthalocyanine are distributed in the mitochondria within tumor cells and induce apoptosis of tumor cells by means of photodamage of apoptosis suppressor protein Bcl-2 [9,10,12,15,18,22,23,33,34—38,42—47]. On the other hand, talaporfin sodium is primarily distributed in the lysosome within tumor cells and does not induce photodamage of Bcl-2 during PDT using this photosensitizer [45]. Furthermore, it has been shown that cells with excessive Bcl-2 expression resisted PDT. In view of these findings, it seems likely that the cytotoxic mechanism of talaporfin sodium used for PDT differs totally from that of photofrin and other photosensitizers [44—47]. It is known that during PDT using lysosomal photosensitizers like talaporfin sodium, cytochrome C is released from mitochondria and pro-caspase-3 is activated [45]. Stolka et al. [47] proposed a mechanism for the release of cytochrome C from the mitochondria, suggesting that conversion of Bid (a Bcl2 supergene family member) to t-Bid due to lysosomal photodamage many triggers the release of cytochrome C from the mitochondria. Reiners et al. [46] reported that the release of cytochrome C activates Apaf/pro-caspase-9, resulting in progression of the apoptosis cascade beginning with pro-caspase-3. In the present study, clear changes in cell morphology such as loss of cell processes, swelling of the cytoplasm and foamy changes began in the deep layer of the tumor mass 1 h after PDT. The nuclei tended to be condensed, although it was difficult to view this change as a typical sign of apoptosis. However, the expression of M30 cytodeath, an indicator of the fragmentation of intermediate diameter filament (cytokeratin 18) due to caspase, in the cytoplasm (i.e., a sign of cell death) suggests that caspasedependent apoptosis of tumor cells was induced [34]. The tumor cell density decreased over time after PDT, and M30 cytodeath expression in tumor cells cytoplasm increased, indicating shrinkage of tumor tissue [34]. It is noteworthy that at 6 h after PDT, the expression of M30 cytodeath was noted also in the tumor cells at the front of the invading area, allowing confirmation that the effect of PDT increased over time. Regarding the transient edema observed in surrounding brain tissue, we noted that blood vessels in the normal brain

208 tissue near the tumor-invading front showed preservation of blood brain barrier (BBB), especially in the rat model of transplanted brain tumor [49]. If this finding is together with the fact that talaporfin sodium does not pass the BBB [28,29], we cannot rule out the possibility that talaporfin sodium leaks out of tumor bulk into surrounding brain tissue. Talaporfin sodium has a high affinity for albumin [30]. It is therefore possible that this compound accumulates in the extracellular matrix of surrounding brain tissue at an early phase after administration (the phase with high plasma drug level). It is highly probable that PDT induces toxic effects in surrounding brain tissue due to formation of singlet oxygen. However, considering that the edematous change ameliorated over time and that no cell damage was observed in the surrounding brain tissue in the present study, it seems highly probable that any talaporfin sodium leaking out into the extracellular matrix was washed out over time. For this reason, edema of surrounding intact brain tissue may be prevented if laser irradiation is applied at a point when the administered talaporfin sodium has been largely washed out from plasma. However, as pointed out by Katsumi et al. [33], the effects of PDT depend on the tissue photosensitizer level as well as on the conditions for irradiation with the diode laser. It is essential to identify the best timing to achieve both high tumor drug level and considerable washout of the drug from plasma. The talaporfin sodium dose level and the conditions for diode laser irradiation are also important [13,19,20,29]. A key point in the success or failure in utilization of talaporfin sodium-PDT as a means of treating malignant glioma is how to achieve efficient formation of singlet oxygen in the target tumor tissue, while avoiding affects on normal brain tissue and blood vessels [41,48].

Conclusion PDT applied using talaporfin sodium in rats transplanted with C6 glioma induced coagulation necrosis and apoptosis of tumor cells. The efficacy of this therapy increased over time, inducing apoptosis even in the tumors cells at the invading area. It remains unresolved whether the apoptosis was triggered by hypoxia associated with vascular shut-down or by the cytotoxic effect of singlet oxygen. The transient edematous change observed in the surrounding brain tissue indicate the essentiality determining the best conditions for PDT, with adequate care paid to prevention of adverse effects on normal blood vessels.

H. Namatame et al.

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The authors are indebted to Professor J.P. Barron of Tokyo Medical University for his review of the manuscript.

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