molecular, cellular, and tissue responses following photodynamic therapy

Lasers in Surgery and Medicine 8:450-463 (1988)

Molecular, Cellular, and Tissue Responses Following Photodynamic Therapy

Charles J. Gomer, PhD, Angela Ferrario, PhD, Naganobu Hayashi, MD, PhD, Natalie Rucker, BS, Bernard C. Szirth, PAV, and A. Linn Murphree, MD

Clayton Ocular Oncology Center, Children’s Hospital of Los Angeles (C.J.G., A.F., N. H., N. R., B.C.S., A. L. M.) and Departments of Pediatrics (Division of Hematology-Oncology) (C. J. G.), Radiation Oncology (C. J.G.), Ophthalmology (A. L. M.), USC School of Medicine,

Los Angeles

Photodynamic therapy (PDT) is being utilized in the treatment of a wide variety of malignant tumors. Results using PDT have been encouraging, and controlled clinical trials are currently being performed. The procedure exploits both the tumor-localizing and -photosensitizing properties of hematoporphyrin derivative or its purified component, Photofrin 11. When this porphyrin mixture is administered systemi- cally, it is retained preferentially in tumor tissue as compared to sur- rounding normal tissue. Localized tumor destruction induced by PDT results from the photochemical generation of cytotoxic oxygen species within the tumor. This review will provide a summary of historical and current research pertaining to molecular, cellular, and tissue responses induced by PDT. Emphasis is placed on information related to the chemistry of current photosensitizers, subcellular targets, preclinical treatment parameters, and clinical responses following PDT.

Key words: hematoporphyrin derivative, laser, photosensitization, tumor

INTRODUCTION

The initial biomedical use of porphyrins was in the area of tumor diagnosis. Hematoporphyrin (Hp) was shown to be taken up in tumor tissue (following systemic administration), and these lesions could then be visualized by fluorescence when exposed to violet light [l]. An improved tumor localizer containing a mixture of monomeric and aggregated porphyrins was produced in 1961 by first acetylating and then reducing the crude hematoporphyrin [ 21. This mixture exhibited more reproducible tumor-localizing properties than Hp and was called hematoporphyrin derivative (HpD). HpD fluorescence as a diagnostic tool was exten- sively examined in the late 1960s and early 1970s 131. The current availability of image intensifiers and endoscopes have played a major role in the successful use of porphyrins in the diagnosis of sputum-positive but X-ray-negative carcinoma in situ of the bronchus [4).

The documentation in the early 1970s that porphyrins were efficient photosensitizers of tumor tissue initiated the current interest in the field of photodynamic therapy [5,6]. Interestingly,

there was little chemical information available regarding the active localizing and photosensitizing components of HpD even though this mixture was used in numerous basic and clinical studies during the 1970s and 1980s. The acetylation procedure utilized in the production of HpD yields several species of porphyrins including monoacetates and diacetates of hematoporphyrin as well as hydroxyethylvinyldeuteroporphyrin and proto- porphyrin (71. When the monoacetates and diacetates of Hp are treated with sodium hydroxide, a polymeric fraction is formed, which constitutes the active material of HpD [8]. The monomeric porphyrins that are found in HpD are not efficient in vivo tumor photosensitizers 171. In the early 1980s, Dougherty separated HpD utilizing gel exclusion chromatography and observed that approximately 45% of the initial HpD was responsible for the

Accepted for publication June 22, 1988. Address reprint requests to Dr. Charles J. Gomer, Clayton Ocular Oncology Center, Children’s Hospital of Los Angeles, 4650 Sunset Boulevard, Los Angeles, CA 90027.

0 1988 Alan R. Liss, Inc.

Responses to Photodynamic Therapy 451

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drug's tumor-photosensitizing properties [9]. The active fraction of HpD is now thought to be an aggregated mixture of hematoporphyrin molecules linked by either ether or ester bonds, as shown in Figure 1 [10,11]. Photofrin I1 is the com- mercial name for the di-hematoporphyrin ether andor ester (DHE) material. This drug is currently available from Quadra Logic Technologies, Inc. Vancouver, British Columbia.

Porphyrins such as HpD and Photofrin I1 possess absorption spectra that make them excellent in vitro photosensitizers, but they are only moderately effective in vivo photosensitizers. These chromophores have an intense absorption in the violet region around 400 nm (termed the soret band) and four additional absorption bands (with decreasing intensity) between 500 and 630 nm [ 121. The evaluation of porphyrin photosensitization in solutions and in cells can utilize light throughout the visible light spectrum. Unfortu- nately, in vivo and clinical photodynamic therapy (PDT) must utilize wavelengths of light corresponding to the weakest porphyrin absorption band. Attenuation of light in tissue decreases with increasing wavelengths [ 131, and therefore light at 630 nm is normally used in PDT. Optical pene- tration depths in tumor tissue (defined as the depth at which light intensity is reduced to 37% of its initial value) are typically less than 1 mm for wavelengths of light below 600 nm and approximately 2.5-4.5 mm for light at 630 nm [14]. The soret absorption band (370-410 nm) is routinely utilized only to activate porphyrin fluorescence for tumor diagnostic studies. The next generation of photosensitizers will undoubtedly possess a major absorption peak at a wavelength above 650 nm. These properties will provide improved penetra- tion depth of delivered light as well as enhanced photon absorption by the drug. Absorption spectra for two new photosensitizers (from the chlorin and phthalocyanine families) are shown in Figure 2 along with the corresponding spectrum for DHE.

Porphyrin photosensitizers can undergo several photochemical reactions. Cellular and tissue damage induced by PDT is believed to result from the formation of singlet oxygen (l02) via the photochemical type I1 reaction shown in Figure 3 [15]. The absorption of a photon of light will excite the porphyrin molecule to an excited and short-lived (lop9 to seconds) singlet state. From the excited singlet state, the photosensitizer can decay back to the ground state and emit light in the form of fluorescence. Kinetic studies have demonstrated that most effective photodynamic sensitiz-

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ers (including porphyrins) will undergo intersystem crossing from the excited singlet state to the more stable and longer-lived seconds) triplet state of the molecule. The excited triplet state has a high probability of interacting with ground state oxygen. The transfer of energy from the triplet photosensitizer to ground state oxygen generates highly reactive singlet oxygen [ 161. Sin- glet oxygen is electrophilic and reacts with electron-rich regions of biomolecules to produce oxidized forms of the molecules [ 171. However, the excited triplet state of the porphyrin molecule can

452 Gomer et a1

also react directly with a biomolecule of interest a role in inducing tolerance to PDT or in cellular by electron or hydrogen atom transfer to produce repair following oxidative stress. radical forms of the sensitizer and substrate. Once While a large spectrum of specific types of formed, the porphyrin radical can interact with subcellular damage has been documented follow- oxygen in a reaction that regenerates the ground ing PDT, the actual target site(s> for cytotoxicity state of the porphyrin molecule and produces hy- have not been satisfactorily identified. In fact, the drogen peroxide, hydroxyl radical, or superoxide targets for in vitro PDT-induced cytotoxicity will [18-201. The actual biological significance of spe- probably vary as a function of the porphyrin incu- cies other than singlet oxygen and hydroxyl radi- bation conditions utilized in individual exper- cals in PDT is still unclear. However, the absolute iments. Short porphyrin incubation time periods requirement for oxygen in the photosensitizing (less than 1 hour) followed by light exposure leads action of clinically used porphyrins has been doc- primarily to plasma membrane damage, whereas umented in solution, in culture, and in vivo [21- extended time periods of porphyrin incubation 23). These results suggest that extremely hypoxic (which may more closely mimic the clinical situa- tumor tissue may be resistant to the direct effects tion) followed by light exposure induces damage of PDT. However, if the tumor vasculature is ac- to cytoplasmic organelles [34]. Standard mamma- tually a primary target in PDT (as suggested by lian cell culture studies have demonstrated that several recent investigations), then the degree of in vitro PDT is neither mutagenic nor carcino- tumor hypoxia at the time of treatment may not genic [27,35]. In addition, porphyrin PDT has been play a major role in the effectiveness of this found to induce comparable levels of cytotoxicity modality. in normal human fibroblasts and in DNA repair-

deficient fibroblasts from ataxia telangiectasia and

IN VITRO PORPHYRIN PHOTOBIOLOGY xeroderma pigmentosum patients [36]. These results provide evidence that nuclear damage andl

Numerous studies have demonstrated that or repair is not a dominant factor in the cytotoxic- PDT is extremely effective in generating cytotoxic ity induced by porphyrin photosensitization. damage to subcellular organelles and biomole- Several basic parameters related to por- cules [ 17,241. Photodegradation of lipids, proteins, phyrin-induced photosensitization of mammalian and nucleic acids can routinely be observed follow- cells have been described. The quantitative depen- ing porphyrin photosensitization. Unsaturated dence of oxygen for porphyrin-induced photoinac- fatty acids can be photooxidized by a singlet oxy- tivation of mammalian cells has been examined gen type I1 mechanism. Lipid peroxidation as well by several investigators. A 1% oxygen concentra- as protein crosslinking of membranes have been tion reduced the quantum yield of photoinactiva- observed in mammalian cells following photosen- tion by approximately 50%, and anoxic cells were sitization. Membrane damage can then lead to found to be resistant to porphyrin photosensitiza- inhibited membrane transport of amino acids and tion [21,221. In addition, normal mouse skin ren- nucleosides, increased permeability and rupture dered anoxic by clamping is resistant to photo- of lysosomes, as well as marked contraction and dynamic therapy [23]. These results indicate that rupture of mitochondria [25]. At the level of the areas of hypoxia may be resistant to direct PDT- nucleus, PDT can produce single-strand breaks induced photosensitization. and alkali-labile sites in DNA, sister chromatid The expression and repair of potentially le- exchanges, and chromosome aberrations [26-281. thal damage can be observed in mammalian cells Enzymes bound to hydrophobic regions of mito- treated with porphyrin photodynamic therapy chondria (membrane-bound) are more sensitive to [37]. Hypothermic exposure (4°C) or incubation of PDT than enzymes located in the hydrophilic mi- PDT-treated cells with caffeine can potentiate the tochondrial matrix [29]. Interestingly, cytosol en- lethal effects of PDT. Recovery from potentially zymes including lactate dehydrogenase, pyruvate lethal damage expressed by post-treatment hypo- kinase, and glucose phosphate isomerase are only thermia is complete within 1 hour of treatment, moderately sensitive to PDT [30]. However, both while recovery from potentially lethal damage ex- RNA-dependent DNA polymerase and DNA-de- pressed by post-treatment caffeine exposure re- pendent RNA polymerase can be inactivated by quires time periods of up to 24 hours. Classical HpD-induced photosensitization [31,32]. Recently, split-dose studies have demonstrated that cells PDT has been shown to activate a variety of cel- treated with PDT can repair sublethal damage lular stress proteins 1331. These proteins may play 1381. However, in vitro and in vivo studies indicate


that the efficiency of porphyrin photosensitization is not effected by nonthermal variations in dose rates of delivered light 1391. Clonigenic assays using Chinese hamster cells have shown that maxi- mal photosensitization utilizing clinically relevant red light is obtained within the narrow range of 630-6325 nm [40]. The action spectra for cell killing matches the absorption spectra for porphyrin bound to cells rather than the absorption spectrum of porphyrin dissolved in aqueous solution (which is blue shifted). Similar action spectra have been reported for in vivo inactivation of tumors as well as for PDT-induced normal skin damage [7].

Several studies have indicated that malignant and normal cells accumulate similar levels of porphyrin during in vitro incubation [41]. In addition, differences in cell photosensitivity are usually not observed for normal and transformed cell lines [41]. However, significant variations in cellular photosensitivity have recently been observed when components of the vasculature were examined [36]. Specifically, bovine cells of endothelial, smooth muscle, and fibroblast origin were compared for porphyrin retention, toxicity, and photosensitization. Bovine endothelial cells were considerably more sensitive than smooth muscle or fibroblast cells treated under identical conditions when assayed for viability using clonigenic- ity. All bovine cells accumulated similar levels of porphyrin, and therefore the increased sensitivity of the endothelial cells was not due to differences in porphyrin retention. These results indicate that endothelial cell photosensitivity may play a role in the vascular damage observed following porphyrin photodynamic therapy.

To begin examining the molecular basis for the observed differential photosensitivity of cellular components of the vasculature, we performed a kinetic analysis of stress protein synthesis following isoeffect doses of PDT. PDT doses that induced 10% survival (315, 420, and 525 J/m2 for endothelial, fibroblast, and smooth muscle cells, respectively) were delivered to cells incubated for 16 hours with DHE. Control cells as well as cells treated with DHE alone, light alone, or hyperthermia were also analyzed. Following treatment, the cells were incubated at 37°C in complete growth medium for designated time intervals (0-12 hours) and then incubated for 2 hours in minimum essen- tial medium (MEM) media supplemented with 35S- methionine. The cells were then collected, and ali- quots were taken for determination of radioactivity, protein concentration, and cell number. Samples were then analyzed by sodium dodecyl

sulfate (SDS) polyacrylamide gel electrophoresis with each lane being loaded with equal levels of radioactivity. Autoradiographs obtained from these gels are shown in Figure 4. Kinetic evaluations of protein synthesis are shown in Figure 5 and were obtained using densitometry readings of specific proteins from the autoradiographs. Con- trols as well as light alone samples had comparable protein gel patterns. Treatment with heat induced a characteristic increase in the 70,000 MW heat shock protein (HSP-70), indicating that all three cell lines were capable of inducing this stress protein. Interestingly, all three cell lines also demonstrated induction of a 34,000 MW protein (presumably a stress protein) when the cells were incubated with DHE in the absence of light as well as after PDT. Smooth muscle and fibroblasts demonstrated a large increase in the amounts of HSP-70 (peaking at about 6-8 hours post-treatment). A 78,000 MW glucose regulated protein (GRP-78) was also induced at extended time periods after treatment for these two cell lines. However, the protein patterns for the most sensitive cell type (endothelial cells) demonstrated only minimal levels of HSP-70 protein production. The significance of these findings is currently unclear. However, HSP-70 is believed to play a role in the induction of cellular tolerance and/or repair.

PORPHYRIN PHOTOCHEMISTRY

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Fig. 3. Type I1 (energy transfer) photochemistry involved in the cytotoxic action of PDT.

454 Gomer et al

BOVINE FIBROBLAST CELLS BOVINE ENDOTHELIAL CELLS

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DHE: 25 UGIML; 16 HR HEAT: 15 MIN. ; 45 C PDT: 420 JOULES/SQ M

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C PDT: 315 JOULESSQ M

Fig. 4. Patterns of protein synthesis in exponentially growing fibroblasts (A), smooth muscle (B), and endothelial cells (C) of bovine origin. Cells were exposed to either control incubation conditions (MEM media supplemented with 10% or 5% fetal calf serum); Photofrin I1 (DHE) alone (16-hour incubation with a drug concentration of 25 pg/ml in MEM media supplemented with 5 8 FCS, followed by a 30-minute washout incubation in fresh MEM media containing 10% FCS); light exposure alone (630 Jim'); heat (15 minutes at 45°C) or PDT (25 p g DHE/ml and a light dose that produced 10% clonogenic survival [see 361 At selected time intervals following treatment, cells were pulse-labeled for 2 hours with 75SS-methionine. Lysates of cells containing equal levels of radioactivity were size-separated using polyacrylamide gel electrophoresis. Resulting gels were stained with Coomassie blue and dried. Autoradiographs of the gels are presented with molecular weight markers shown on the left and the major stress proteins induced by heat and/or PDT shown on

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IN VIVO AND PRECLINICAL PHOTODYNAMIC THERAPY 3 1 0 -

Interest in porphyrin PDT for the treatment of solid tumors started in the early 1970s. Initial work in 1972 showed that Hp combined with white light induced extensive damage to glioma tumors

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transplanted in rats [ 5 I . Long-term cures were reported in 1975 when HpD, combined with red light, was used to treat spontaneous and transplanted


mammary carcinomas in mice 161. The initial studies from Dougherty's group at Roswell Park Memorial Institute (Buffalo, NY) have had a major impact on the extensive growth and utilization of PDT during the last 10 years [7].

Pharmacokinetics of HpD have been analyzed utilizing radiolabeled materials (14C-HpD and 3H-HpD) in tumor-bearing mice [42]. Hema- toporphyrin derivative and subsequently Pho- tofrin I1 have been shown to be retained in tumor tissue a t higher concentrations than in normal tissues such as skin, muscle, brain, and lungs. However, significantly greater quantities of these porphyrins are taken up and retained in organs such as liver, spleen, and kidney. While the actual mechanism for the delivery and retention of porphyrins in tumor tissues is not known, recent re- ports indicate that there is a high porphyrid lipoprotein affinity [43,44]. Porphyrins such as HpD and Photofrin I1 can bind to various lipoprotein fractions in the serum including low-density lipoproteins (LDL). It is interesting to note that the number of LDL receptor sites is elevated in neoplastic cells as well as in the various organs in which there are relatively high concentrations of porphyrin. In agreement with these observations is the report that there is a significant enhance- ment in the preferential uptake of Hp in tumor tissue when Hp is bound to LDL prior to systemic administration in tumor-bearing mice [45].

While the photochemical properties of photodynamic therapy indicate that singlet oxygen is a primary agent in the induction of tumoricidal effects, the actual targets of PDT at the tissue level have still not been clearly defined. A number of studies have indicated that both normal and tumor vasculature can be severely damaged following photodynamic therapy. Sophisticated in vivo/ in vitro studies have demonstrated that the cloni- genicity of EMT-6 and RIF tumor cells is not reduced if the cells are taken from treated mouse tumors immediately following PDT treatments that would normally lead to significant cytotoxicity if the tumors were left in situ [46]. In separate studies, blood flow was reduced to almost zero in transplantable rat bladder tumors following PDT [47]. Taken together, these and other studies suggest that vascular damage plays an important role in the mechanism of tumor response following PDT.

Experiments were performed to determine if PDT could destroy experimentally induced ocular blood vessels [48]. For these experiments, corneal neovascularization was induced in albino rabbits using paracentral silk sutures with exposed knots.

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Fig. 5. Kinetics of stress protein synthesis as a function of time following isoeffect doses of PDT. Densitometer readings were obtained from scanning the autoradiographs described in Figure 4 and are ratios of (A) 78-kD stress proteins (GRP- 78) vs. actin, (B) 70-kD stress proteins (HSP-70) vs. actin; and (C) 34-kD stress proteins vs. actin. Ratios for fibroblasts (+I, smooth muscle (21, and endothelial cells (0 ) are shown for each stress protein.

Using this method, discrete corneal vessels ex- tending a n average of 3.5 mm into the clear cornea could be created. The model of neovascularization provided an excellent method for visualizing both the vessels and the effect of PDT on these vessels. Initial treatments utilized intravenously (1.V.) in-

Gomer et al

n.. LIGHT DELIVERI PLAQUE

Fig. 6. The ability of porphyrin photosensitization to destroy blood vessels is exploited in trans-scleral PDT for the treatment of experimental choroidal melanoma. A trans-scleral light delivery probe (emitting unidirectional light over a 0.78- cm2 area) is placed on the sclera. Light is delivered through the sclera and into the highly vascularized choroid. The sclera does not take up porphyrin, while the choroid contains high concentrations of porphyrin following I.V. administration. All blood and nutrients supplied to choroidal melanomas come from the choroidal vessels directly under the scleral probe.

jected HpD (5 mgkg). At various time periods following injection individual quadrants of vascularized cornea were exposed to either krypton (407 nm), argon (488 and 514 nm), or dye laser (630 nm) light. Pre- and post-treatment fluorescein an- giograms were performed to document the vascular integrity of the treated vessels. These initial studies indicated that PDT could induce a regres- sion in the neovascularization. Histological examination was performed 48 hours after treatment in select animals, and these studies revealed destruction of the vascular endothelium. Violet light, which is absorbed more efficiently by porphyrins than red light, induced complete vascular destruction at lower total light doses than that required for treatment with red light. Dose-response experiments and histopathological evaluations are currently being completed.

As described above, there is considerable evidence that PDT can effectively destroy blood vessels in both tumor tissue and normal tissue. This phenomenon is currently being exploited in the preclinical evaluation of trans-scleral PDT for the treatment of choroidal melanomas. The concept of trans-scleral light delivery for ocular PDT is at- tractive for several reasons (Fig. 6). First, HpD pharmacology data indicates that the choroid contains high levels of HpD immediately after I.V.

injection 1491. Of equal importance is the fact that the sclera does not take up significant amounts of HpD. A second attraction of trans-scleral light delivery is that porphyrin-activating red light can be delivered precisely and selectively to specific areas of the vascular choroid. This structure sup- plies all oxygen and nutrients to choroidal melanoma.

Preliminary experiments have been performed using pigmented rabbits to determine the effectiveness of trans-scleral PDT for destroying a select area of the choroid. Initial results indicate that this procedure can produce complete and localized choroidal damage with vascular occlusion. Rabbits were injected I.V. with Photofrin I1 (5 mg/ kg followed either 1 hour or 24 hours later by localized trans-scleral PDT. The diameter of the light delivery probe was 10 mm, and the dose of 630-nm light was 200 J/cm2. The light dose rate utilized in these experiments was 100 mW/cm2. Complete choroidal and retinal necrosis was observed in the treatment field, while areas outside the light delivery field appeared normal. Of equal importance in these preliminary studies was the observation that the sclera within the treatment field was not damaged as documented by light microscopy and visual examination. The lack of scleral damage was expected since we have observed and reported that HpD is not localized in this structure. Control eyes, which received light alone, appeared normal both inside and outside of the treatment field. PDT treatments initiated soon after porphyrin injection were significantly more effective as compared to those initiated 24 hours after injection.

The examination of hypoxic cell cytotoxic agents combined with PDT has been initiated as a result of the observed vascular damage and resulting induction of hypoxic tumor tissue following PDT. Enhancements in tumor growth delays have been obtained in rat prostate tumors treated with a combination of PDT and misonidazole, and this potentiation is thought to result from direct misonidazole-induced cytotoxicity of hypoxic cells [50,51]. These observations indicate that hypoxic cell cytotoxins could be utilized to enhance the effectiveness of PDT. However, while vascular effects of PDT continue to be documented, there are also in vivo studies indicating that direct tumor cell inactivation via PDT can be exploited. Murine ascites tumors have been effectively treated with interperitoneal U.P.) PDT 1521. Tumor cures reach- ing a n 85% level have been obtained utilizing a total of 4 PDT treatments given at 2-day intervals


in C3H mice containing ascitic malignant tera- toma cells. Treatment of the same tumor with chemotherapy (adriamycin) induced less than 20% cures.

Examination of tumor metastasis induced by PDT was evaluated because this treatment is being suggested for use in early-stage clinical ma- lignancies and because of the continued observation of direct vascular injury following PDT [53]. Studies were performed to determine whether localized PDT treatment of subcutaneously growing Lewis lung carcinoma influenced the formation of distant metastasis. Mice exposed to localized PDT treatment had significantly reduced numbers of metastatic lung colonies compared to controls. In addition, PDT-treated mice had equal or decreased numbers of metastatic lung colonies than comparable mice treated with local surgery. These results indicate that local PDT does not enhance the metastatic spread of Lewis lung carcinoma following treatment.

Recent investigations have examined various immunological responses following PDT. This treatment has been shown to induce systemic immunosuppression related to inhibition of contact hypersensitivity of dinitrofluorobenzene in mice [ 541. The immunosuppression induced by PDT may be mediated by the activation of the complement system 155, 561. We have recently examined the natural killer (NK) cell activity in non-tumor- bearing mice treated with localized photodynamic therapy. A correlation has been reported between NK cell activity and the in vivo capacity to elimi-

PDT: Melanotic Melanoma

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Fig. 7. Tumor response curves for C57BU6 mice implanted with a B-16 melanotic melanoma and treated with PDT. Tu- mors (5-7 mm in diameter) were exposed to 630-nm light 24 hours following a 10 mgKg I.P. injection of Photofrin 11. Total light doses ranged from 0 to 500 J/cm*, and light dose rates were 150 (P), 300 (+), 450 (01, or 600 (0) m (W/crnZ. Percent cure was determined from animals that were disease-free 45 days following treatment. Each point represents the average of ten animals.

nate circulating tumor cells [57j. There is also increasing evidence to suggest that NK cells play a significant role in the resistance to progressive growth and metastasis of tumors 1581. It was therefore of interest to determine the effect of PDT on NK cell activity and to see whether this response correlated with the lack of metastatic induction observed with PDT 1531. C57BL/6 mice were used to evaluate NK cell activity as well as to document spleen weight following porphyrin administration and light exposure. Splenic NK cell activity was determined utilizing the procedure of Pollock et al. [59]. The YAC-1 lymphoma cell line was labeled with 5'Cr sodium chromate

PDT: AMELANOTIC MELANOMA

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MELANOTIC MELANOMA, 630 NM, 2 mm DEPTH

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Fig. 9. B-16 melanotic melanoma tumor temperature as a function of time during 630-nm light exposure. Light was delivered at dose rates of 150 (E), 300 (+), 450, (E) or 600 (0) mW/cm2. The treatment spot diameter was 1 cm, and temperatures were measured at a 2-mm depth from the tumor s ~ - face using a 21-gauge thermocouple hypodermic needle (Omega). Each point represents the average of five tumors.

458 Gomer et a1

TABLE 1. Natural Killer Cell Activity and Spleen Weight in C57RL16 Mice Following Photofrin I1 Administration

Spleen weight

Percent cell lysis at Days following effector: target ratioa photofrin I 1 Photofrin I1 No. administration dose (mgkg) of mice 1OO: l 50:l 25:l (mg)

1 0 5 24.2 17.4 11.8 60 10 4 22.0 16.2 11.0 65 50 4 18.5 13.0 8.0 69.2

3 0 8 23.3 17.0 11.6 65 10 7 19.2 13.6 8.8 75.8 50 7 19.2 14.4 9.5 92.8

10 7 28.4 20.4 13.1 87.8 50 7 27.4 16.1 10.7 106.5

14 0 4 21.5 16.2 9.7 50.2 10 7 25.0 20.0 13.2 82 50 4 15.0 11.5 7.0 75.2

7 0 7 26.1 18.7 12.2 55.4

"Effector cells were spleen cells; target cells were 2 x lo4 "Cr-labeled YAC-1 cells.

TABLE 2. Natural Killer Cell Activity and Spleen Weight in C57BL/6 Mice Following - Photodynamic Therapy*

Days following Percent cell lysis at Spleen photodynamic No. of effector: target ratio" . . weight

1 Control 7 20.5 18.8 10.0 73.5 PDT 10 3.7 2.5 1.2 40.3

3 Control 6 28.0 22.6 12.8 96.5 PDT 8 21.2 17.5 9.1 55

7 Cont.rol 4 30.0 22.7 12.2 63.5 PDT 8 26.6 21.6 10.6 88.1

14 Control 5 24.7 17.7 8.2 81 ._ - PDT 13 29.5 23.9 11.3 83

*Photofrin I1 (10 mgikg) administered 24 hours prior to PDT. PDT consisted of a 400 Jlcm' dose delivered over a 1-cm-diameter area of the right hindleg at a dose rate of 150 mW/cm2. "Effector cells were spleen cells; target cells were 2 x lo4 51Cr-labeled YAC-1 cells.

t h e r a p y . . Treatment . - mice 1OO:l 50:l 25:l (mg)..

and was then used as the target cell in NK assays. Splenic effector cells at various dilution ratios were incubated with 2 x lo4 YAC cells. Photofrin I1 alone at concentrations of 10 and 25 mgkg induced a significant increase in spleen size as indicated in Table 1. The maximum increase in spleen weight was observed 7 days following porphyrin administration. While the spleen size in mice injected with Photofrin I1 increased, there was no change from control values for NK cell activities. However, alterations in both NK cell activity and in spleen weight were observed for mice treated with PDT, and results are shown in Table 2. PDT consisted of a 10 mgkg I.P. dose of Photofrin I1 followed 24 hours later by local expo-

sure of the right hindlimb to 400 J/cm2 of 630-nm light. A rapid decrease in both spleen size and NK cell activity was observed following PDT. Within 24 hours, the NK cell activity was reduced to levels near zero. Associated with this decrease in NK activity was a reduction in spleen weight to approximately one-half of control values. Both of these changes were transient in nature, with NK cell activity and spleen size returning to control values within 3 days.

The observed decrease in NK cell activity following PDT appears to be a systemic effect, since only the hindlimb of the experimental animal received the light treatment. The observed decrease in NK activity could be the results of


prostaglandin release, which has been shown in the past to effect NK cell activity. Therefore, while PDT does not enhance metastatic spread in experimental animal models [53], it does appear that this modality can induce immunosuppression related to both T-cells (documented in hypersensitivity reactions) and NK cells.

Hyperthermia in the range of 42.5-47"C can potentiate the tumoricidal action of ionizing radiation as well as numerous chemotherapeutic agents. Studies performed during the past few years indicate that a synergistic interaction also exists between hyperthermia and PDT [SO]. In vitro and in vivo experiments have demonstrated that potentiation of PDT response by hyperthermia depends on the sequence of treatments, the time interval between treatments, and the temperature used. Maximum potentiation appears to occur with temperatures above 40.5"C and when heat is applied immediately following PDT.

High-intensity arc lamps appropriately fil- tered to emit a broad spectrum of red light were used as initial light exposures in preclinical and clinical PDT treatments. The continued exploita- tion of PDT has been greatly enhanced by the utilization of argon pumped dye lasers, which can generate high-intensity monochromatic light. Quartz fibers are efficiently interfaced to dye lasers so that porphyrin-activating light can be delivered to various tumor sites. The distal ends of the fibers can be manipulated with diffusers or lenses for interstitial insertion into subcutaneous or pul- monary lesions, for cylindrical light delivery to the lumen for esophageal tumors, for 360 degree delivery to bladder lesions, and for forward delivery to cutaneous or ocular tumors [7,14].

A comprehensive set of experiments was recently performed to evaluate the relationship between in vivo PDT tumor response and dose rate (150-600 mW/cm2) of delivered light. Comple- menting this study was the documentation of tumor temperature rise as a function of light dose rate. Two strains of B-16 melanoma (one pigmented and one nonpigmented) transplanted to the flank of C57BL/6 mice were utilized to generate PDT tumor response curves (Figs. 7,8). Tumor size at the time of treatment (5-7 mm diameter), DHE dose (10 mgkg), and treatment spot size (1 cm diameter) were all kept constant. If reciprocity was observed, as was the case in previous in vitro experiments and in vivo normal tissue PDT experiments erformed with light dose rates below 150 mW/cm [39], then a single tumor response curve would have been generated. However, the tumor

E

response curves shift to the left with increasing light dose rate. The primary cause for the shift in the tumor response curves is shown in Figures 9 and 10 where tumor temperature is recorded for each light dose rate. Temperature rises (at a 2-mm depth in each tumor) ranged from 5" to 25°C. It is clear that these temperature levels played a major role since tumor treatments performed without DHE (data not shown) produced identical tumor response data when light dose rates of 450 and 600 mW/cm2 were used. A synergistic response of PDT and heat (4045°C) has also been reported, and therefore light dose rates in the range of 150- 300 mW/cm2 (which do not produce significant levels of tumor cures by themselves) may enhance the tumoricidal effects of PDT. It should be noted that normal tissue effects of PDT will also be enhanced by hyperthermia, so there may not be an increase in the therapeutic ratio (tumor vs. normal tissue response) when increased light dose rates are used.

Preclinical studies designed to evaluate the efficacy of PDT have been performed for anatomical sites including the bronchus, eye, and bladder. Dogs with carcinogen-induced squamous cell carcinomas of the bronchus were utilized to evaluate PDT treatments in which light was delivered through a fiber in a biopsy channel of a broncho- scope [61]. Complete tumor responses were documented histologically in animals injected with HpD (2.5-3.0 mgkg) 48 hours prior to the delivery of red light (60-240 J/cm2). Bronchial tumors in animals receiving light alone or drug alone had no histological changes. Pigmented rabbits were used in ocular toxicity experiments in the initial evaluation of PDT for the treatment of intraocular tumors 149, 621. An intravenous injection of HpD was followed 48 hours later by exposure of a 1-cm2 area of the retina to transpupillary delivered red light. The light was generated by an argon pumped dye laser tuned to 630 nm, and toxicity to normal ocular structures was documented by fundus pho- tography, fluorescein angiography, and histological evaluation. Acute ocular toxicity in the form of retinal adema, detachment, and necrosis was induced by clinically relevant dosages of PDT. However, the area of ocular damage was limited to the treatment field for all but the highest doses of PDT. Histological results indicated that there was an abrupt and demarcated area between in- jured and normal-appearing retina. Long-term toxicity (9- to 30-month follow-up period) demonstrated that the retinal damage that was induced in the treatment field was permanent but not pro-

Gomer et al

AMELANOTIC MELANOMA, 630 NM. 2 mm DEPTH 30 7 I

I 20

10

0 0 1 0 2 0 3 0

TIME (minutes)

Fig. 10. B-16 amelanotic melanoma tumor temperature rise as a function of time during PDT light exposure. Other details as per Figure 9.

gressive. Lens opacities were not observed during the follow-up period, and the cornea, aqueous, and vitreous remained clear in all tested animals. The efficacy of PDT has also been examined in normal dog bladders as well as in rabbit bladders transplanted with the Brown-Pearce carcinoma 163- 661. A 360 J/cm2 dose of 630-nm light delivered to a l-cm2 area of normal dog bladder 3 days after a 2.5 mgkg injection of Photofrin I1 produced a lesion that extended to the muscle layer but did not induce a fistula. Light doses less than 360 J/cm2 produced only superficial damage to the exposed area. Results from rabbit experiments demonstrated that HpD was preferentially taken up in the transplanted bladder carcinoma and that PDT could effectively destroy the tumor without damage to normal urothelium.

CLINICAL PDT

The first use of prophyrin PDT for the clinical treatment of solid tumors was in 1976, and currently more than 3,000 patients have received this therapy [7,14,24]. The only significant side effect associated with PDT has been transient skin photosensitization. For some patients, the skin sensitivity can last more than 6 weeks after systemic injection of HpD or Photofrin I1 as a result of prolonged retention of the porphyrin in the skin. While there are anecdotal cases of PDT for almost every anatomical site and histological type of solid cancer, the use of PDT appears to be most encouraging for solid tumors of the bronchus, esophagus, bladder, head and neck, skin, and eye. As with most new cancer therapies, the majority of patients treated with PDT have been advanced cases in whom all conventional therapies have failed, and unfortunately there is little information related to long-term response. Early-stage lesions (for which there are significantly fewer patients)

routinely have excellent initial tumor responses following PDT. Several extensive reviews on clinical PDT have been published [7,14], and therefore only recent clinical highlights will be presented.

PDT appears to be effective in superficial bladder cancer, and PDT may find an important role in the management of diffuse carcinoma in situ and in noninvasive transitional cell carcinoma [67]. Bladder tumors can be treated with PDT by delivering light directly into the bladder via an endoscope. Individual lesions can be exposed to unidirectional light emitted from a flat- tipped fiber, while a diffusing bulb can be used for whole-bladder illumination. An excellent correlation between HpD localization and histologically defined dysplasia and carcinoma in situ of the bladder has been reported [66]. In addition, PDT has been extremely effective against Ta and T1 disease for lesions less than 1 cm, but PDT has not been effective for lesions greater than 1.5 cm [68].

Obstructive endobronchial tumors are treated with PDT by using a fiberoptic broncho- scope. Good tumor responses and improvements in patient performance status are routinely observed provided that a clean-up bronchoscopy is performed 2-3 days following PDT [69,70]. These lesions can receive PDT using either surface tumor illumination or by inserting a diffusing cylindrical fiber directly into the lesion for interstitial PDT (711. The use of PDT for obstructive esophageal tumors utilizes techniques similar to that used for endobronchial tumors. The early response for esophageal lesions are encouraging, and the procedure appears to offer excellent palliation [72].

Primary and metastatic skin lesions have re- sponded favorably to PDT [ 141. Metastatic breast carcinomas recurrent to the chest wall can be treated with PDT using either surface or interstitial light delivery. Multiple PDT treatments are also possible. The primary problem in treating skin tumors has been to minimize PDT-induced normal skin toxicity. Basal cell carcinomas appear to be extremely sensitive to PDT, and this treatment has been used successfully in patients with nevoid basal cell carcinoma syndrome [73].

The optical characteristics of the eye combined with the relatively small size of intraocular tumors suggest that ocular PDT could be extremely useful in the treatment of eye tumors. Transcorneal PDT delivered with high light dose rates (greater than 400 mW/cm2) is effective in reducing the height of pigmented choroidal melanomas. In the absence of pigment, as in the case of retinoblastoma or amelanotic melanoma of the iris or choroid, tumor kill attributed to PDT alone can be significant 1741.


Clinical PDT trials utilizing Photofrin I1 have recently been reinitiated with the assistance of Lederle Laboratories, American Cyanamid and Quadra Logics Tech., Inc. Three major anatomical tumor sites are being evaluated. Non-small cell endobronchial carcinoma will be evaluated in two trials. First, in unilateral disease radiation therapy with or without PDT will be evaluated. The second lung trial will be performed in sympto- matic patients with metastatic disease, and in this case PDT will be compared to Nd:YAG laser therapy. For this study, endpoints will include time to first remission as well as relief of pain. Two trials are also being evaluated for esophageal carcinoma. The first trial will again compare Nd:YAG laser therapy to PDT. Endpoints will be time to first remission. The second study is a single-arm trial for completely obstructive esophageal tumors. Bladder carcinomas will be evaluated in the third group of clinical trials. For carcinoma in situ, PDT will be compared to standard thio-TEPA treatment. In addition, a second trial is planned in which prophylaxis treatment of bladder tumors will be evaluated.

ACKNOWLEDGMENTS

We thank Barbara Paul for assistance in the

This work was supported in part by USPHS preparation of this manuscript.

grants CA-31230, CA-43087, and CA-44733.

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molecular, cellular, and tissue responses following photodynamic therapy

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