Eur Arch Otorhinolaryngol (1992) 249:309-312
European Archives of
Oto-RhinoLaryngology (c~ Springer-Verlag 1992
Mode of action of photodynamic therapy with sulfonated aluminum phthalocyanine in induced squamous cell carcinomas in animal models W. v o n Glafi 1, M.Kfisler 2, *, and 3". Lang 1 1Department of Oto-Rhino-Laryngology, University of Erlangen-Ntirnberg, Erlangen, Federal Republic of Germany 2Department of Head and Neck Surgery, Postgraduate Medical School, University of Budapest, Budapest, Hungary Received September 6, 1991 / Accepted March 17, 1992
S u m m a r y . In order to investigate the mechanism of action of photodynamic therapy (PDT) with sulfonated aluminum phthalocyanine (AISPc) in squamous cell carcinoma, animal experiments were performed in induced carcinomas of the mucosa of the hamster's cheek pouch and skin of the laboratory mouse. Histological examinations revealed signs of massive interstitial bleeding, indicating a vascular response to P D T with A1SPc. It was also possible to induce similar change adjacent to newly formed vessels at the margin of an inflammatory reaction in the cheek pouch of five hamsters in the absence of tumor cells. Implanted human squamous cell carcinoma cells in athymic nude mice showed that carcinoma cells removed immediately following P D T remained viable, while tumors left in situ became necrotic. These resuits suggest that the primary effect of P D T with A1SPc in vivo is not the malignant cell itself, but the vascular stroma of the tumor or in the immediate vicinity of the latter. Key words: Photodynamic therapy - Aluminium phthalocyanine - Induced squamous cell carcinoma implants Laboratory animal models
Introduction The basic principle of photodynamic therapy (PDT) of tumors is the systemic administratoin of a suitable photosensitizer and a time delay to allow selective accumulation of the sensitizer in the tumor, followed by irradiation of the lesion and adjacent tissue. The irradiation employs light at a wavelength at which the sensitizer has an absorption peak, and which at the same time permits maximum tissue penetration. The most commonly inves* Awardee of the Humboldt Fellowship at the ENT Department, University of Erlangen-Ntirnberg Correspondence to: W. von Glag, Paracelsusweg 9, W-7302 Ostfildern 1, Federal Republic of Germany
tigated photosensitizer to date is hematophorphyrin derivative (HpD), a mixture of various porphyrins of which dihematophorphyrin ether has the greatest photodynamic effect [2]. A further substance suitable for use a photosensitizing agent is sulfonated aluminum phthalocyanine (A1SPc) [11. The mode of action of P D T is controversial. In the case of H p D an indirect effect via the vascular system of the tumor has been considered responsible [3, 4, 6, 7]. The present study was undertaken in an attempt to clarify the mode of action of P D T with AISPc in the case of squamous cell carcinoma, one of the most c o m m o n tumor types encountered in the head and neck.
Material and m e t h o d s A n i m a l models In order to induce squamous cell carcinomas, the mucosa of the everted cheek pouches of adult Syrian hamsters (Mesocricetus auratus) and the skin of white mice (Mus musculus) were painted three times a week for 4 weeks with the carcinogen 9,10-dimethyl1,2-benzanthracene at a concentration of 0.5 g/t00 ml acetone [5]. Six months later tumor growth was observed in both cheek pouches of all hamsters and in the skin of 40% of the mice. To produce an inflammatory mucosal reaction, an area measuring roughly 0.4 cm in diameter on both sides of the everted cheek pouch of the hamsters was superficially charred with the aid of bipolar coagulation forceps. PDT at the neoplastic and inflammatory lesions was applied 48 h after creating the inflammatory reaction. In male athymic nude mice (strain Han: NMRI nu/nu) 0.4 cm × 0.2cm pieces of poorly differentiated human squamous cell carcinoma from the larynx were implanted subcutaneously bilaterally just above the hind legs. The tumor originated in a 42-year-old patient and prior to implantation had undergone a single passage through nude mice, with a take rate of about 89%. PDT was carried out when the implanted tumor had attained a size of at least 0.5 cm in diameter. Laser The laser employed was an argon-pumped MDS 83 dye laser (Aesculap-Meditec, Heroldsberg). The light of the laser was delivered
310
OO
a
mine (Ketanest) and 16mg/kg xylacine (Rompun). Twenty-four hours prior to photoirradiation, the animals received 10mg/kg A1SPc via intraperitoneal injection. Thereafter, an area measuring 1.0 cm in diameter was irradiated with light at a wavelength of 677 nm for 6 min and 15 s at a power fluence density of 0.4 W/cm 2 resulting in an energy fluence density of 150J/cm 2. Immediately prior to and following irradiation, temperature was determined with the aid of a quick response thermometer containing a 1-mmdiameter sensor (Testoterm 9500; Testoterm GmbH, Lenzkirch). Immediately after the laser treatment, and 24h thereafter, excisional biopsies were obtained for histology from hamsters' cheek pouch carcinomas and the skin carcinomas of the mice. This allowed sequential observations of any further reactions of the tumor treatment. An inflammatory mucosal reaction in the cheek pouch of the hamster was sampled for histological examination both immediately after treatment with the laser and 24 h later. In the case of the nude mice the tumors on each leg were treated separately with the laser. Immediately thereafter, the last treated tumour was removed, while the other was left in situ. From the tumor removed, two 0.4cm x 0.2 cm fragments were taken from the superficial portion receiving the greatest amount of light during laser treatment, and reimplanted in the region of the shoulder of the same mouse (Fig. 1). The subsequent behavior of the tumor left in situ and the reimplants were then followed closely.
Results
PDT of mucosal tumors in hamsters C
d
PDT was performed in eight animals on one tumor of one cheek pouch. Immediately after laser treatment, the exophytic papillomatous, well-perfused, rosy tumors turned dark red to blue-black. One day after PDT, the irradiated tumor started to become necrotic. Non-irradiated control tumors of the opposite cheek pouch remained viable, while the concurrently irradiated mucosa also remained macroscopically unchanged. Within the next few days, the completely necrotic laser-treated tumors sloughed. In the tissue biopsies obtained after PDT, histological findings regularly demonstrated diffuse, intestitial bleeding into the tumor stroma (Fig. 2). If they were at all detectable, capillary blood vessels were often filled with erythrocytes. Often, the entire tumor stroma was also pervaded by many erythrocytes. Prior to laser irradiation, the average temperature mea-
Fig. l a - d . Schematic representation of transplantation experiments with human squamous cell carcinomas in athymic nude mice during photodynamic therapy, a Two fragments of a human squamous cell carcinoma are implanted into the experimental animal, b The implants have grown to a minimum size of 0.5cm in diameter. c Both implants are treated with laser light 24 h after administration of photodynamic sensitizer, d One irradiated tumor is left in situ, while the other is exl~lanted. Fragments of the explanted tumor are reimplanted at another site
via a flexible quartz fiber which was position-adjusted with the aid of a micromainpulator. The effective power emitted from the tip of fiber was measured with a power meter prior to each application.
Experimental protocol All procedures performed on the animals were carried out under anesthesia induced by intramuscular injections of 100 mg/kg keta-
Fig. 2. Histology of a squamous cell carcinoma in the hamster cheek pouch mucosa following photodynamic therapy with A1SPc: the stroma is pervaded with erythrocytes H & E stain, x 350
31l sured in the superficial parts of the tumor was 29.2°C. After irradiation the average temperature had increased to 32.5°C (with an average difference of 3.3°C).
P D T of skin carcinomas in mice P D T was carried out in seven tumors. Immediately after laser application, no unequivocal changes could be observed in the exulcerating tumors. One day later, parts of the tumor was grossly unchanged, while other parts showed superficial sloughing. After 4-5 days, the irradiated tumor area was found to be partly necrotic, with the extent of necrosis varying considerably from case to case. However, in the floor and at the margins of tumor, apparently viable carcinoma tissue was to be seen. In the biopsies obtained after PDT, histologly revealed interstital bleeding wherever a well-developed vascular stroma was found. In the biopsies obtained 1 day following laser irradiation, the superficial parts of the tumor were often partially necrotic. Otherwise there were not significant differences when tissue was compared with the excisional biopsies taken on the previous day. The average temperature measured at the surface of the skin carcinomas was 30.0°C prior to laser application and 32.0°C after irradiation (average difference, 2.0°C).
PD T of carcinomas transplanted to nude mice P D T and retransplantation experiments were performed in nine nude mice. Immediately after laser application, in situ tumor revealed no visible changes. Three to 4 days later, the skin over the tumor had turned livid and then later became necrotic. A partical necrosis of the superficial three-quarters occurred, with basal parts of the carcinoma remaining viable in seven animals and unchecked tumor growth occurring in two. Following reimplantation, a take rate of 86% was observed. The average temperature of the skin over the transplanted tumors was 34. I°C prior to irradiation and 35.2°C after irradiation (average difference 1.1°C).
P D T of inflammatory mucosal reactions in the hamster As a result of coagulation, the inflamed mucosa was converted to a whitish scab which 2 days later was suppurating. On the surface of the coagulation necrosis, numerous newly formed blood vessels were found arranged in a concentric manner, greatly ramifying towards the margin of the necrosis. P D T of these lesions was carried out in five animals. Immediately following laser application, a diffuse reddish tinge was observed around the lesion and was due to bleeding into the mucosa. Occasionally, circumscribed bluish-colored hematomas were also seen. In the cheek pouches removed immediately following PDT, histological evaluation revealed that the vessels formed at the margin of the inflammatory reaction were completely filled with erythrocytes. In additon, regularly diffuse interstitial hemorrhages were also detectable. These changes were located in the floor and at the rim of the ulcer. The mucosa at some distance from the inflammatory reaction remained unremarkable. The changes
seen in the cheek pouch removed 1 day after laser treatment showed no significant differences from those removed immediately.
Discussion
The most commonly employed sensitizer used in P D T at the present time is HpD. The tumor necrosis induced by P D T using H p D is due to vascular effects [3, 4, 6, 7]. A further substance with a photodynamic effect is A1SPc, but is mode of action is unclear. In the present investigation, an attempt was made to establish whether in the P D T of squamous cell carcinoma using A1SPc, effects on the blood circulation similar to those achievable with H p D can be found, and whether such effects suffice to explain tumor destruction. Among the carcinomas induced in the hamster cheek pouch, P D T with A1SPc led to a grossly recognizable striking bluish-black discoloration of the rosy pre-treatment tumor appearance, reflecting the presence of diffuse interstitial bleeding in the tumor stroma. The area of tumor treatment became completely necrotic within a matter of days, indicating a high level of efficacy of P D T with A1SPc. In the irradiated parts of the squamous cell carcinomas of the skin of the mouse, necrosis also occurred. However, in contrast to the mucosal carcinomas in the hamster, no such impressive growth effect was observed immediately after laser treatment. This can be explained by our findings that the mouse carcinomas were appreciably less vascular when compared with the hamster tumors. Wherever the mouse carcinomas were more vascular, interstitial bleeds were again readily detected. The results of our present experiments demonstrate that P D T with A1SPc as sensitizer leads to vascular effects in tumor stroma, while no such phenomena are triggered in normal tissue. The question now arises as to whether this selectivity is due solely to particular circumstances within the stroma or in the blood vessels present or whether the malignant tumor cells themselves, at least in part, are also indirectly responsible. In order to exclude the latter possibility, we then considered whether similar vascular effects - that is, interstitial bleeding as found in tumor stroma could also be triggered under certain conditions without the presence of tumor cells. For this purpose, a circumscribed area of mucosa of the hamster cheek pouch was destroyed by the application of heat to induce an inflammatory reaction. A proliferative tissue reaction was then produced comprising the formation of connective tissue and tiny new blood vessels. In the region of these newly formed blood vessels, P D T with A1SPs triggered bleeding into the mucosa that was of sufficient intensity to be grossly recognizable. Histologically, these bleeds were identical with those found in the tumor stroma. These findings show that vascular effects can be produced by P D T with AISPc, irrespective of whether a malignant tumor is present or not. However, we also realize that this observation does not exclude the possibility that P D T has an additional effect on the malignant tumor cell itself.
312 In order to investigate possible t u m o r effects of P D T in squamous cell carcinomas, two carcinoma implants of defined sizes were placed subcutaneously in athymic nude mice and concurrently subjected to photoradiation. One of the tumors was left in situ and kept under observation, while the other was explanted immediately after PDT. Superficial fragments of the explanted t u m o r were then reimplanted subcutaneously in the same mouse in the region of the shoulder not receiving laser treatment. The tumors left in situ developed necrosis in their superficial portions after P D T with either sensitizer. Thus, for reimplantation, those parts of the t u m o r were employed which developed necrosis in. the tumors left in situ. Nevertheless, most of these carcinoma cells r e m o v e d immediately following P D T and transplanted into nude mice continued to grow. Following retransplantation, a take rate was obtained which was almost equal to the rate prior to PDT. On the basis of these results we conclude that squamous cell carcinoma cells r e m o v e d immediately after P D T with A1SPc are viable, while carcinoma cells left in situ after treatment are destroyed. Our present results have permitted us to conclude that P D T of squamous cell carcinomas with A1SPc, even at the high concentrations of photosensitizer and light employed in the experiments described, does not lead directly to t u m o r cell necrosis, but has an indirect effect outside the t u m o r cells. It is certain that the vascular effect produced is of decisive importance for t u m o r necrosis, and this mechanism is probably the only one responsible for t u m o r destruction in our animals. Never-
theless, the possibility has not yet been reliably excluded that P D T results in other mechanisms outside the t u m o r cell that also lead to tumour necrosis.
References 1. Brown SG, Tralau CJ, Colderidge Smith PD, Akdemir D, Wieman TJ (1986) Photodynamic therapy with porphyrin and phthalocyanine sensitisation: quantitative studies in normal rat liver. Br J Cancer 54: 43-52 2. Dougherty TJ, Potter WR, Weishaupt KR (1984) The structure of the active component of hematoporphyrin derivative. In: Dorion DR, Gomer DJ (eds) Porphyrin localization and treatment of tumours. Liss, New York, pp 301-314 3. Glag W yon, K~isler M, Lang T (1991) Experimentelle Untersuchungen zur photodynamischen Therapie von Plattenepithelkarzinomen mit H~imatoporphyrinderivat. HNO 39 : 91-97 4. Henderson BW, Waldow SM, Mang TS, Potter WR, Malone PB, Dougherty TJ (1985) Tumor destruction and kinetics of tumor cell death in two experimental mouse tumors following photodynamic therapy. Cancer Res 45 : 572-576 5. Salley JJ (1954) Experimental carcinogenesis in the cheek pouch of the Syrian hamster. J Dent Res 33 : 253-262 6. Selman SH, Kreimer-Birnbaum M, Klaunig JE, Goldblatt PJ, Keck RW (1984) Blood flow in transplantable bladder tumors with hematoporphyrin derivative and light. Cancer Res 44: 1924-1927 7. Star WM, Marijnissen JPA, Berg-Blok AE van der, Versteeg JAC, Franken KAP, Reinhold HS (1986) Destruction of rat mammary tumor and normal tissue microcirculation by hematoporphyrin derivative phot.oradiation observed in vivo in sandwich observation chambers. Cancer Res 46:2532
European Archives of
Oto-RhinoLaryngology (c~ Springer-Verlag 1992
Mode of action of photodynamic therapy with sulfonated aluminum phthalocyanine in induced squamous cell carcinomas in animal models W. v o n Glafi 1, M.Kfisler 2, *, and 3". Lang 1 1Department of Oto-Rhino-Laryngology, University of Erlangen-Ntirnberg, Erlangen, Federal Republic of Germany 2Department of Head and Neck Surgery, Postgraduate Medical School, University of Budapest, Budapest, Hungary Received September 6, 1991 / Accepted March 17, 1992
S u m m a r y . In order to investigate the mechanism of action of photodynamic therapy (PDT) with sulfonated aluminum phthalocyanine (AISPc) in squamous cell carcinoma, animal experiments were performed in induced carcinomas of the mucosa of the hamster's cheek pouch and skin of the laboratory mouse. Histological examinations revealed signs of massive interstitial bleeding, indicating a vascular response to P D T with A1SPc. It was also possible to induce similar change adjacent to newly formed vessels at the margin of an inflammatory reaction in the cheek pouch of five hamsters in the absence of tumor cells. Implanted human squamous cell carcinoma cells in athymic nude mice showed that carcinoma cells removed immediately following P D T remained viable, while tumors left in situ became necrotic. These resuits suggest that the primary effect of P D T with A1SPc in vivo is not the malignant cell itself, but the vascular stroma of the tumor or in the immediate vicinity of the latter. Key words: Photodynamic therapy - Aluminium phthalocyanine - Induced squamous cell carcinoma implants Laboratory animal models
Introduction The basic principle of photodynamic therapy (PDT) of tumors is the systemic administratoin of a suitable photosensitizer and a time delay to allow selective accumulation of the sensitizer in the tumor, followed by irradiation of the lesion and adjacent tissue. The irradiation employs light at a wavelength at which the sensitizer has an absorption peak, and which at the same time permits maximum tissue penetration. The most commonly inves* Awardee of the Humboldt Fellowship at the ENT Department, University of Erlangen-Ntirnberg Correspondence to: W. von Glag, Paracelsusweg 9, W-7302 Ostfildern 1, Federal Republic of Germany
tigated photosensitizer to date is hematophorphyrin derivative (HpD), a mixture of various porphyrins of which dihematophorphyrin ether has the greatest photodynamic effect [2]. A further substance suitable for use a photosensitizing agent is sulfonated aluminum phthalocyanine (A1SPc) [11. The mode of action of P D T is controversial. In the case of H p D an indirect effect via the vascular system of the tumor has been considered responsible [3, 4, 6, 7]. The present study was undertaken in an attempt to clarify the mode of action of P D T with AISPc in the case of squamous cell carcinoma, one of the most c o m m o n tumor types encountered in the head and neck.
Material and m e t h o d s A n i m a l models In order to induce squamous cell carcinomas, the mucosa of the everted cheek pouches of adult Syrian hamsters (Mesocricetus auratus) and the skin of white mice (Mus musculus) were painted three times a week for 4 weeks with the carcinogen 9,10-dimethyl1,2-benzanthracene at a concentration of 0.5 g/t00 ml acetone [5]. Six months later tumor growth was observed in both cheek pouches of all hamsters and in the skin of 40% of the mice. To produce an inflammatory mucosal reaction, an area measuring roughly 0.4 cm in diameter on both sides of the everted cheek pouch of the hamsters was superficially charred with the aid of bipolar coagulation forceps. PDT at the neoplastic and inflammatory lesions was applied 48 h after creating the inflammatory reaction. In male athymic nude mice (strain Han: NMRI nu/nu) 0.4 cm × 0.2cm pieces of poorly differentiated human squamous cell carcinoma from the larynx were implanted subcutaneously bilaterally just above the hind legs. The tumor originated in a 42-year-old patient and prior to implantation had undergone a single passage through nude mice, with a take rate of about 89%. PDT was carried out when the implanted tumor had attained a size of at least 0.5 cm in diameter. Laser The laser employed was an argon-pumped MDS 83 dye laser (Aesculap-Meditec, Heroldsberg). The light of the laser was delivered
310
OO
a
mine (Ketanest) and 16mg/kg xylacine (Rompun). Twenty-four hours prior to photoirradiation, the animals received 10mg/kg A1SPc via intraperitoneal injection. Thereafter, an area measuring 1.0 cm in diameter was irradiated with light at a wavelength of 677 nm for 6 min and 15 s at a power fluence density of 0.4 W/cm 2 resulting in an energy fluence density of 150J/cm 2. Immediately prior to and following irradiation, temperature was determined with the aid of a quick response thermometer containing a 1-mmdiameter sensor (Testoterm 9500; Testoterm GmbH, Lenzkirch). Immediately after the laser treatment, and 24h thereafter, excisional biopsies were obtained for histology from hamsters' cheek pouch carcinomas and the skin carcinomas of the mice. This allowed sequential observations of any further reactions of the tumor treatment. An inflammatory mucosal reaction in the cheek pouch of the hamster was sampled for histological examination both immediately after treatment with the laser and 24 h later. In the case of the nude mice the tumors on each leg were treated separately with the laser. Immediately thereafter, the last treated tumour was removed, while the other was left in situ. From the tumor removed, two 0.4cm x 0.2 cm fragments were taken from the superficial portion receiving the greatest amount of light during laser treatment, and reimplanted in the region of the shoulder of the same mouse (Fig. 1). The subsequent behavior of the tumor left in situ and the reimplants were then followed closely.
Results
PDT of mucosal tumors in hamsters C
d
PDT was performed in eight animals on one tumor of one cheek pouch. Immediately after laser treatment, the exophytic papillomatous, well-perfused, rosy tumors turned dark red to blue-black. One day after PDT, the irradiated tumor started to become necrotic. Non-irradiated control tumors of the opposite cheek pouch remained viable, while the concurrently irradiated mucosa also remained macroscopically unchanged. Within the next few days, the completely necrotic laser-treated tumors sloughed. In the tissue biopsies obtained after PDT, histological findings regularly demonstrated diffuse, intestitial bleeding into the tumor stroma (Fig. 2). If they were at all detectable, capillary blood vessels were often filled with erythrocytes. Often, the entire tumor stroma was also pervaded by many erythrocytes. Prior to laser irradiation, the average temperature mea-
Fig. l a - d . Schematic representation of transplantation experiments with human squamous cell carcinomas in athymic nude mice during photodynamic therapy, a Two fragments of a human squamous cell carcinoma are implanted into the experimental animal, b The implants have grown to a minimum size of 0.5cm in diameter. c Both implants are treated with laser light 24 h after administration of photodynamic sensitizer, d One irradiated tumor is left in situ, while the other is exl~lanted. Fragments of the explanted tumor are reimplanted at another site
via a flexible quartz fiber which was position-adjusted with the aid of a micromainpulator. The effective power emitted from the tip of fiber was measured with a power meter prior to each application.
Experimental protocol All procedures performed on the animals were carried out under anesthesia induced by intramuscular injections of 100 mg/kg keta-
Fig. 2. Histology of a squamous cell carcinoma in the hamster cheek pouch mucosa following photodynamic therapy with A1SPc: the stroma is pervaded with erythrocytes H & E stain, x 350
31l sured in the superficial parts of the tumor was 29.2°C. After irradiation the average temperature had increased to 32.5°C (with an average difference of 3.3°C).
P D T of skin carcinomas in mice P D T was carried out in seven tumors. Immediately after laser application, no unequivocal changes could be observed in the exulcerating tumors. One day later, parts of the tumor was grossly unchanged, while other parts showed superficial sloughing. After 4-5 days, the irradiated tumor area was found to be partly necrotic, with the extent of necrosis varying considerably from case to case. However, in the floor and at the margins of tumor, apparently viable carcinoma tissue was to be seen. In the biopsies obtained after PDT, histologly revealed interstital bleeding wherever a well-developed vascular stroma was found. In the biopsies obtained 1 day following laser irradiation, the superficial parts of the tumor were often partially necrotic. Otherwise there were not significant differences when tissue was compared with the excisional biopsies taken on the previous day. The average temperature measured at the surface of the skin carcinomas was 30.0°C prior to laser application and 32.0°C after irradiation (average difference, 2.0°C).
PD T of carcinomas transplanted to nude mice P D T and retransplantation experiments were performed in nine nude mice. Immediately after laser application, in situ tumor revealed no visible changes. Three to 4 days later, the skin over the tumor had turned livid and then later became necrotic. A partical necrosis of the superficial three-quarters occurred, with basal parts of the carcinoma remaining viable in seven animals and unchecked tumor growth occurring in two. Following reimplantation, a take rate of 86% was observed. The average temperature of the skin over the transplanted tumors was 34. I°C prior to irradiation and 35.2°C after irradiation (average difference 1.1°C).
P D T of inflammatory mucosal reactions in the hamster As a result of coagulation, the inflamed mucosa was converted to a whitish scab which 2 days later was suppurating. On the surface of the coagulation necrosis, numerous newly formed blood vessels were found arranged in a concentric manner, greatly ramifying towards the margin of the necrosis. P D T of these lesions was carried out in five animals. Immediately following laser application, a diffuse reddish tinge was observed around the lesion and was due to bleeding into the mucosa. Occasionally, circumscribed bluish-colored hematomas were also seen. In the cheek pouches removed immediately following PDT, histological evaluation revealed that the vessels formed at the margin of the inflammatory reaction were completely filled with erythrocytes. In additon, regularly diffuse interstitial hemorrhages were also detectable. These changes were located in the floor and at the rim of the ulcer. The mucosa at some distance from the inflammatory reaction remained unremarkable. The changes
seen in the cheek pouch removed 1 day after laser treatment showed no significant differences from those removed immediately.
Discussion
The most commonly employed sensitizer used in P D T at the present time is HpD. The tumor necrosis induced by P D T using H p D is due to vascular effects [3, 4, 6, 7]. A further substance with a photodynamic effect is A1SPc, but is mode of action is unclear. In the present investigation, an attempt was made to establish whether in the P D T of squamous cell carcinoma using A1SPc, effects on the blood circulation similar to those achievable with H p D can be found, and whether such effects suffice to explain tumor destruction. Among the carcinomas induced in the hamster cheek pouch, P D T with A1SPc led to a grossly recognizable striking bluish-black discoloration of the rosy pre-treatment tumor appearance, reflecting the presence of diffuse interstitial bleeding in the tumor stroma. The area of tumor treatment became completely necrotic within a matter of days, indicating a high level of efficacy of P D T with A1SPc. In the irradiated parts of the squamous cell carcinomas of the skin of the mouse, necrosis also occurred. However, in contrast to the mucosal carcinomas in the hamster, no such impressive growth effect was observed immediately after laser treatment. This can be explained by our findings that the mouse carcinomas were appreciably less vascular when compared with the hamster tumors. Wherever the mouse carcinomas were more vascular, interstitial bleeds were again readily detected. The results of our present experiments demonstrate that P D T with A1SPc as sensitizer leads to vascular effects in tumor stroma, while no such phenomena are triggered in normal tissue. The question now arises as to whether this selectivity is due solely to particular circumstances within the stroma or in the blood vessels present or whether the malignant tumor cells themselves, at least in part, are also indirectly responsible. In order to exclude the latter possibility, we then considered whether similar vascular effects - that is, interstitial bleeding as found in tumor stroma could also be triggered under certain conditions without the presence of tumor cells. For this purpose, a circumscribed area of mucosa of the hamster cheek pouch was destroyed by the application of heat to induce an inflammatory reaction. A proliferative tissue reaction was then produced comprising the formation of connective tissue and tiny new blood vessels. In the region of these newly formed blood vessels, P D T with A1SPs triggered bleeding into the mucosa that was of sufficient intensity to be grossly recognizable. Histologically, these bleeds were identical with those found in the tumor stroma. These findings show that vascular effects can be produced by P D T with AISPc, irrespective of whether a malignant tumor is present or not. However, we also realize that this observation does not exclude the possibility that P D T has an additional effect on the malignant tumor cell itself.
312 In order to investigate possible t u m o r effects of P D T in squamous cell carcinomas, two carcinoma implants of defined sizes were placed subcutaneously in athymic nude mice and concurrently subjected to photoradiation. One of the tumors was left in situ and kept under observation, while the other was explanted immediately after PDT. Superficial fragments of the explanted t u m o r were then reimplanted subcutaneously in the same mouse in the region of the shoulder not receiving laser treatment. The tumors left in situ developed necrosis in their superficial portions after P D T with either sensitizer. Thus, for reimplantation, those parts of the t u m o r were employed which developed necrosis in. the tumors left in situ. Nevertheless, most of these carcinoma cells r e m o v e d immediately following P D T and transplanted into nude mice continued to grow. Following retransplantation, a take rate was obtained which was almost equal to the rate prior to PDT. On the basis of these results we conclude that squamous cell carcinoma cells r e m o v e d immediately after P D T with A1SPc are viable, while carcinoma cells left in situ after treatment are destroyed. Our present results have permitted us to conclude that P D T of squamous cell carcinomas with A1SPc, even at the high concentrations of photosensitizer and light employed in the experiments described, does not lead directly to t u m o r cell necrosis, but has an indirect effect outside the t u m o r cells. It is certain that the vascular effect produced is of decisive importance for t u m o r necrosis, and this mechanism is probably the only one responsible for t u m o r destruction in our animals. Never-
theless, the possibility has not yet been reliably excluded that P D T results in other mechanisms outside the t u m o r cell that also lead to tumour necrosis.
References 1. Brown SG, Tralau CJ, Colderidge Smith PD, Akdemir D, Wieman TJ (1986) Photodynamic therapy with porphyrin and phthalocyanine sensitisation: quantitative studies in normal rat liver. Br J Cancer 54: 43-52 2. Dougherty TJ, Potter WR, Weishaupt KR (1984) The structure of the active component of hematoporphyrin derivative. In: Dorion DR, Gomer DJ (eds) Porphyrin localization and treatment of tumours. Liss, New York, pp 301-314 3. Glag W yon, K~isler M, Lang T (1991) Experimentelle Untersuchungen zur photodynamischen Therapie von Plattenepithelkarzinomen mit H~imatoporphyrinderivat. HNO 39 : 91-97 4. Henderson BW, Waldow SM, Mang TS, Potter WR, Malone PB, Dougherty TJ (1985) Tumor destruction and kinetics of tumor cell death in two experimental mouse tumors following photodynamic therapy. Cancer Res 45 : 572-576 5. Salley JJ (1954) Experimental carcinogenesis in the cheek pouch of the Syrian hamster. J Dent Res 33 : 253-262 6. Selman SH, Kreimer-Birnbaum M, Klaunig JE, Goldblatt PJ, Keck RW (1984) Blood flow in transplantable bladder tumors with hematoporphyrin derivative and light. Cancer Res 44: 1924-1927 7. Star WM, Marijnissen JPA, Berg-Blok AE van der, Versteeg JAC, Franken KAP, Reinhold HS (1986) Destruction of rat mammary tumor and normal tissue microcirculation by hematoporphyrin derivative phot.oradiation observed in vivo in sandwich observation chambers. Cancer Res 46:2532
