204
3 E. Ben-Hur, R. Kol, R. Marko, E. RikIis and I. Rosenthal, Combined action of phthalocyanine photosensitization and gamma-irradiation on mammalian cells, Int. J. Radiat. Biol., 54 (1988) 2130. 4 S. P. Creekmore and D. S. Zaharto, Modification of chemotherapeutic effects on L1210 cells using hematoporphyrin derivative and light, Cancer Res., 43 (1983) 5252-5257. 5 J. S. Nelson, L.-H. Liaw, R. A. Lahlum, P. L. Cooper and M. W. Berns, Use of multiple photosensitixers and wavelengths during photodynamic therapy: a new approach to enhance tumor eradication, J. N&Z. Cancer Inst., 82 (1990) 868-873. 6 W. C. Beckman, S. K. Powers, J. T. Brown, G. Y. Gillespie, D. D. Bigmer and J. L. Camps, Differential retention of Rh123 by avian sarcoma virus-induced gIioma and normal brain tissue of the rat in vivo, Cancer, 59 (1987) 266-270. 7 H. Tapiero, J.-N. Munck, A. Fourcade and T. J. Lampidis, Cross-resistance to rhodamine 123 in adriamycin- and daunorubicln-resistant Friend leukemia cell variants, Cancer Res., 44 (1984) 5544-5549. 8 A. A. Neyfahk, Use of fluorescent dyes as molecular probes for the study of multidrug resistance, Exp. CeU. Res., 174 (1988) 168-176. 9 D. Kessel, Exploring multidrug resistance using rhodamine 123, Cancer Commun., 1 (1989) 145-149. 10 V. Ling and J. Gerlach, Multidrug resistance, Breast Cancer Res. Treatment, 4 (1985) 89-94. 11 G. Singh, B. C. Wilson, S. M. Sharkey, G. P. Browman and P. Des&s, Resistance to photodynamic therapy in radiation induced fibrosarcoma-1 and Chinese hamster ovary multidrug resistant cells in vitro, Photo&em. Photobtil., 54 (1991) 307-312. 12 D. Kessel and C. Erickson, Porphyrin photosensitization of multidrug resistant celI types, Photo&em. Photobiol., in the press.
The chick chorioallantoic membrane (CAM) as an in wiwo model for photodynamic therapy V. Gottfried”, E. S. Lindenbaumb and S. Kimel” aDe_vartment of Chemistry, bMorphology Unit, FacuUy Institute of Technology, Haifa 32000 Qsrael)
of Medicine,
Technion,
Israel
Photodynamic therapy (PDT) involves, as a primary step, damage either directly to the tumor cells or to the surrounding vasculature, which causes disruption of tumor blood flow and tissue necrosis by anoxia [ 1, 21. The chick chorioallantoic membrane (CAM) model has been adapted for the study of the vascular damage occurring in PDT [ 3 1. The CAM is composed of three components: the outer ectoderm, consisting of a single layer of cells; the middle mesoderm, composed of 2-3 layers of connective-tissue cells and fibers in which the capillaries are located; the inner endoderm, consisting of a single layer of cells [4]. Together they form a transparent
205
matrix which allows direct observation of the microcirculation. It is possible to view individual blood vessels and to examine, in real time, structural changes in the vasculature. The CAM provides a viable, economic alternative to laboratory testanimals (e.g. rats, mice, chickens or rabbits) normally used for in viva studies of vascular phenomena during PDT. The limitations associated with light penetration in animal tissue do not pertain to the transparent CAM, so that the “therapeutic” wavelength of choice in CAM-PDT is not limited to the red region of the spectrum. Sensitizers can be applied topically and may be varied to include the clinically employed porphyrins as well as secondgeneration drugs proposed in the literature. The model is uniquely suited for videotaping the extent of PDT-induced injury to the vasculature. This in viva system can also be used as a host for tumor cell transplants [5, 61. Through implantation of different cancer cell lines onto the CAM host this model can replace tumor-bearing animal systems for screening new drugs and for establishing light and drug dose relationships. The model is expected to shed light on one of the major unknowns in the mechanism of PDT: whether it is cellular or vascular [ 1, 21. The protocol for CAM preparation is a modification of a previously described technique [ 71. Fertilized eggs are fumigated, washed with ethanol solution (70 vol.% in distilled water), incubated at 37 “C in 60% humidity, and rolled over hourly. On day 3-4, the apex of the egg is cleaned with 70% ethanol, a hole is drilled through the egg shell, and 2-3 ml of albumin is withdrawn from each egg in order to create a false air sac. On the following day, part of the CAM is exposed by opening a round window of approximately 2 cm diameter in the shell, which is covered with a Petri dish. The incubation is continued (no rolling over) until day 9 when the CAM is fully developed and ready for experimentation. The sensitizer solution is applied directly on the CAM within an area confined by a Teflon ring. Preparation of sensitizer solutions, deposition of rings on the CAM, and application of sensitizer are carried out under sterile conditions to avoid contamination of the CAM. Since photochemotherapeutic agents used in PDT contain fluorescing chromophores, their uptake, localization, and retention can be monitored directly in a transparent medium such as the CAM. On the treatment day, uptake of the sensitizer and its diffusion into the CAM is assayed, after selected time intervals, to allow for the topically applied sensitizer to diffuse into the CAM. The exposed area of the membrane is ilxed for 1 h with 8% formaldehyde, cut out, washed with distilled water, spread on a glass slide, and protected with a cover glass. The fluorescence in the CAM is measured using a fluorescence microscope. This flxed-time technique for monitoring the uptake of sensitizers is complemented by a real-time method for measuring the fluorescence spectrum and intensity in viwo [B]. The CAM surface is coupled to a fluorimeter by means of fibre optics which transmitsthe excitation light from the instrument as well as the emission from the CAM surface to the detection system of the fluorimeter.
PDT is begun on day 9 when the CAM is fully developed. The photosensitizer is applied topically on the CAM and is irradiated with light of the appropriate wavelength. The CAM is inspected blindly (as to sensitizer and light dosages) by independent observers, starting one day after activation of the photosensitizer (to avoid interference of the diagnostic light on the photodynamic process). The damage to blood vessels as observed through a stereoscopic microscope, at a magnification of up to 40 X, is quantified and photographed. Alternatively, the photodynamic process is videotaped in real time, which provides a qualitative insight in the PDT mechanistics during the evolution of damage. The CAM versatility as a host has been demonstrated in several systems: tumor growth [ 51, synergy of hyperthermia and PDT [ 81 and synergy of hyperthermia and chemotherapy [91. Tumors grafted on the CAM [ 51 can be used to study differences in uptake between normal (endothelial) cells and tumor cells, as well as to examine the effects of PDT on the implant and on the angiogenic [6] tumor-associated neovasculature. Since the CAM is a time-limited system, existing within a window of time which is between the 9th day of incubation (when the CAM angiogenesis is completed) and the 16th day (when the chick immunological system becomes functional), the fast proliferation characteristics of grafted cells are of prime importance in achieving a working system. The advantages of the CAM bioassay are that it is cost effective and that statistically significant results can be obtained within a relatively short period of time. Moreover, it is available for continuous monitoring in real time and it enables observation of the host vascular reaction to the implant prior to, during and after PDT. The model of grafted cells can be used to study the uptake pattern of different sensitizers in the intact and in the tumor-bearing CAM, using real-time fluorescence and fixed-time fluorescence microscopy. Also, cellular and vascular response of the intact and tumorbearing CAM to photodynamic therapy can be analyzed using light microscopy and histology.
1 D. Kessel (ed.), Photodynamic Therapy of Neoplustic Disease, Vols. I and II, CRC Press, Boca Raton, FL, 1990. 2 C. N. Zhou, Mechanismsof tumor necrosis induced by photodynamic therapy,J. Photo&em. Photobiol., B: Biology, 3 (1989) 299318. 3 V. Gottfried, E. S. Lindenbaum and S. Kimel, Vascular damage during PDT as monitored in the chick chorioallantoic membrane, Iti. J. &&at. Bill., 60 (1991) 349-354. 4 A. Fuchs and E. S. Lindenbaum, The two- and three-dimensional structure of the chick chorloallantoic membrane, Acta Anut., 131 (1988) 271-275. 5 J. Leighton, The spread of cancer explored in the embryonated egg. In The Spread of Cancer, Academic Press, New York, 1967, Chapter 7, pp. 1X-192. 6 J. Folkman, Tumor anglogenesis, Adv. Cancer Res., 43 (1986) 173-203. 7 D. Knighton, D. Ausprunk, D. Tapper and J. Folkman, Avascular and vascular phases of tumor growth in the chick embryo, Br. J. Cancer, 35 (1977) 347-366.
207 8 S. Kimel, L. 0. Svaasand, M. Hammer-Wilson, V. Gottfried and M. W. Bems, The chick chorioallantoic membrane for the study of synergistic effects of hyperthermia and photodynamic therapy, Proc. SPL??,1525, in the press. 9 T. Uchibayashi, M. Egawa, T. Asari, K. Nakqjima, H. Hisazumi, Y. Endo, M. Tanaka and T. Sasaki, Responses of tumor cell lines and surgical specimens transplanted into chorioallantoic membrane of chick embryo to anticancer agents in combination with/without hyperthermia, Jpn. J. UrnroSy, 80 (1989) 17-21.
3 E. Ben-Hur, R. Kol, R. Marko, E. RikIis and I. Rosenthal, Combined action of phthalocyanine photosensitization and gamma-irradiation on mammalian cells, Int. J. Radiat. Biol., 54 (1988) 2130. 4 S. P. Creekmore and D. S. Zaharto, Modification of chemotherapeutic effects on L1210 cells using hematoporphyrin derivative and light, Cancer Res., 43 (1983) 5252-5257. 5 J. S. Nelson, L.-H. Liaw, R. A. Lahlum, P. L. Cooper and M. W. Berns, Use of multiple photosensitixers and wavelengths during photodynamic therapy: a new approach to enhance tumor eradication, J. N&Z. Cancer Inst., 82 (1990) 868-873. 6 W. C. Beckman, S. K. Powers, J. T. Brown, G. Y. Gillespie, D. D. Bigmer and J. L. Camps, Differential retention of Rh123 by avian sarcoma virus-induced gIioma and normal brain tissue of the rat in vivo, Cancer, 59 (1987) 266-270. 7 H. Tapiero, J.-N. Munck, A. Fourcade and T. J. Lampidis, Cross-resistance to rhodamine 123 in adriamycin- and daunorubicln-resistant Friend leukemia cell variants, Cancer Res., 44 (1984) 5544-5549. 8 A. A. Neyfahk, Use of fluorescent dyes as molecular probes for the study of multidrug resistance, Exp. CeU. Res., 174 (1988) 168-176. 9 D. Kessel, Exploring multidrug resistance using rhodamine 123, Cancer Commun., 1 (1989) 145-149. 10 V. Ling and J. Gerlach, Multidrug resistance, Breast Cancer Res. Treatment, 4 (1985) 89-94. 11 G. Singh, B. C. Wilson, S. M. Sharkey, G. P. Browman and P. Des&s, Resistance to photodynamic therapy in radiation induced fibrosarcoma-1 and Chinese hamster ovary multidrug resistant cells in vitro, Photo&em. Photobtil., 54 (1991) 307-312. 12 D. Kessel and C. Erickson, Porphyrin photosensitization of multidrug resistant celI types, Photo&em. Photobiol., in the press.
The chick chorioallantoic membrane (CAM) as an in wiwo model for photodynamic therapy V. Gottfried”, E. S. Lindenbaumb and S. Kimel” aDe_vartment of Chemistry, bMorphology Unit, FacuUy Institute of Technology, Haifa 32000 Qsrael)
of Medicine,
Technion,
Israel
Photodynamic therapy (PDT) involves, as a primary step, damage either directly to the tumor cells or to the surrounding vasculature, which causes disruption of tumor blood flow and tissue necrosis by anoxia [ 1, 21. The chick chorioallantoic membrane (CAM) model has been adapted for the study of the vascular damage occurring in PDT [ 3 1. The CAM is composed of three components: the outer ectoderm, consisting of a single layer of cells; the middle mesoderm, composed of 2-3 layers of connective-tissue cells and fibers in which the capillaries are located; the inner endoderm, consisting of a single layer of cells [4]. Together they form a transparent
205
matrix which allows direct observation of the microcirculation. It is possible to view individual blood vessels and to examine, in real time, structural changes in the vasculature. The CAM provides a viable, economic alternative to laboratory testanimals (e.g. rats, mice, chickens or rabbits) normally used for in viva studies of vascular phenomena during PDT. The limitations associated with light penetration in animal tissue do not pertain to the transparent CAM, so that the “therapeutic” wavelength of choice in CAM-PDT is not limited to the red region of the spectrum. Sensitizers can be applied topically and may be varied to include the clinically employed porphyrins as well as secondgeneration drugs proposed in the literature. The model is uniquely suited for videotaping the extent of PDT-induced injury to the vasculature. This in viva system can also be used as a host for tumor cell transplants [5, 61. Through implantation of different cancer cell lines onto the CAM host this model can replace tumor-bearing animal systems for screening new drugs and for establishing light and drug dose relationships. The model is expected to shed light on one of the major unknowns in the mechanism of PDT: whether it is cellular or vascular [ 1, 21. The protocol for CAM preparation is a modification of a previously described technique [ 71. Fertilized eggs are fumigated, washed with ethanol solution (70 vol.% in distilled water), incubated at 37 “C in 60% humidity, and rolled over hourly. On day 3-4, the apex of the egg is cleaned with 70% ethanol, a hole is drilled through the egg shell, and 2-3 ml of albumin is withdrawn from each egg in order to create a false air sac. On the following day, part of the CAM is exposed by opening a round window of approximately 2 cm diameter in the shell, which is covered with a Petri dish. The incubation is continued (no rolling over) until day 9 when the CAM is fully developed and ready for experimentation. The sensitizer solution is applied directly on the CAM within an area confined by a Teflon ring. Preparation of sensitizer solutions, deposition of rings on the CAM, and application of sensitizer are carried out under sterile conditions to avoid contamination of the CAM. Since photochemotherapeutic agents used in PDT contain fluorescing chromophores, their uptake, localization, and retention can be monitored directly in a transparent medium such as the CAM. On the treatment day, uptake of the sensitizer and its diffusion into the CAM is assayed, after selected time intervals, to allow for the topically applied sensitizer to diffuse into the CAM. The exposed area of the membrane is ilxed for 1 h with 8% formaldehyde, cut out, washed with distilled water, spread on a glass slide, and protected with a cover glass. The fluorescence in the CAM is measured using a fluorescence microscope. This flxed-time technique for monitoring the uptake of sensitizers is complemented by a real-time method for measuring the fluorescence spectrum and intensity in viwo [B]. The CAM surface is coupled to a fluorimeter by means of fibre optics which transmitsthe excitation light from the instrument as well as the emission from the CAM surface to the detection system of the fluorimeter.
PDT is begun on day 9 when the CAM is fully developed. The photosensitizer is applied topically on the CAM and is irradiated with light of the appropriate wavelength. The CAM is inspected blindly (as to sensitizer and light dosages) by independent observers, starting one day after activation of the photosensitizer (to avoid interference of the diagnostic light on the photodynamic process). The damage to blood vessels as observed through a stereoscopic microscope, at a magnification of up to 40 X, is quantified and photographed. Alternatively, the photodynamic process is videotaped in real time, which provides a qualitative insight in the PDT mechanistics during the evolution of damage. The CAM versatility as a host has been demonstrated in several systems: tumor growth [ 51, synergy of hyperthermia and PDT [ 81 and synergy of hyperthermia and chemotherapy [91. Tumors grafted on the CAM [ 51 can be used to study differences in uptake between normal (endothelial) cells and tumor cells, as well as to examine the effects of PDT on the implant and on the angiogenic [6] tumor-associated neovasculature. Since the CAM is a time-limited system, existing within a window of time which is between the 9th day of incubation (when the CAM angiogenesis is completed) and the 16th day (when the chick immunological system becomes functional), the fast proliferation characteristics of grafted cells are of prime importance in achieving a working system. The advantages of the CAM bioassay are that it is cost effective and that statistically significant results can be obtained within a relatively short period of time. Moreover, it is available for continuous monitoring in real time and it enables observation of the host vascular reaction to the implant prior to, during and after PDT. The model of grafted cells can be used to study the uptake pattern of different sensitizers in the intact and in the tumor-bearing CAM, using real-time fluorescence and fixed-time fluorescence microscopy. Also, cellular and vascular response of the intact and tumorbearing CAM to photodynamic therapy can be analyzed using light microscopy and histology.
1 D. Kessel (ed.), Photodynamic Therapy of Neoplustic Disease, Vols. I and II, CRC Press, Boca Raton, FL, 1990. 2 C. N. Zhou, Mechanismsof tumor necrosis induced by photodynamic therapy,J. Photo&em. Photobiol., B: Biology, 3 (1989) 299318. 3 V. Gottfried, E. S. Lindenbaum and S. Kimel, Vascular damage during PDT as monitored in the chick chorioallantoic membrane, Iti. J. &&at. Bill., 60 (1991) 349-354. 4 A. Fuchs and E. S. Lindenbaum, The two- and three-dimensional structure of the chick chorloallantoic membrane, Acta Anut., 131 (1988) 271-275. 5 J. Leighton, The spread of cancer explored in the embryonated egg. In The Spread of Cancer, Academic Press, New York, 1967, Chapter 7, pp. 1X-192. 6 J. Folkman, Tumor anglogenesis, Adv. Cancer Res., 43 (1986) 173-203. 7 D. Knighton, D. Ausprunk, D. Tapper and J. Folkman, Avascular and vascular phases of tumor growth in the chick embryo, Br. J. Cancer, 35 (1977) 347-366.
207 8 S. Kimel, L. 0. Svaasand, M. Hammer-Wilson, V. Gottfried and M. W. Bems, The chick chorioallantoic membrane for the study of synergistic effects of hyperthermia and photodynamic therapy, Proc. SPL??,1525, in the press. 9 T. Uchibayashi, M. Egawa, T. Asari, K. Nakqjima, H. Hisazumi, Y. Endo, M. Tanaka and T. Sasaki, Responses of tumor cell lines and surgical specimens transplanted into chorioallantoic membrane of chick embryo to anticancer agents in combination with/without hyperthermia, Jpn. J. UrnroSy, 80 (1989) 17-21.
