I N 1972, Diamond, et al., 2 first reported the photodynamic

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1 J Neurosurg 64: , 1986 In vitro photoradiation therapy of the rat 9L gliosarcoma DOUGLAS HAMILTON, M.Sc., JOHN D. S. MCKEAN, M.B.CB.B., F.R.C.S.(C), JOHN TULIP, PH.D., DONALD BOISVERT, M.D., PH.D., AND JUDY CUMMINS Division of Neurosurgery, Faculty of Medicine, and Department of Electrical Engineering, Faculty of Engineering, University of Alberta, Edmonton, Alberta, Canada ~" The authors have investigated various factors involved in the photoradiation treatment of 9L glioma cells. The cells were grown in tissue culture and exposed to light from a laser source that allowed accurate quantitation of the light energy. Cell death was determined following treatment using the trypan blue exclusion test. It was shown that the treatment is very wavelength-dependent following the absorption spectrum of hematoporphyrin derivative (HPD). The absorption peaks in the lower part of the spectrum are more efficient than those of higher wavelengths. Photoradiation therapy is more effective the higher the concentration of HPD. Intensity of light is a very important factor in calculating the total dose of light necessary for this treatment. KEY WORDS ~ photoradiation therapy 9 hematoporphyrin derivative 9 9L glioma 9 laser 9 rat I N 1972, Diamond, et al., 2 first reported the photodynamic destruction of a nervous system tumor. They used the rat 9L glioma cell model in tissue culture, incubating it with hematoporphyrin and then exposing the cells to light from eight 20-W fluorescent cool lamps. The cells were stained with trypan blue to test for survival. They found that 100% cell death could be achieved after 50 minutes' exposure to light after incubation in 10-2 M hematoporphyrin. Control cells kept in the dark showed no destruction. In the in vivo experiment conducted by Diamond, et al., the 9L cells were inoculated into the flank of male Fischer 344 rats. After 19 to 21 days the animals were given 10 mg/kg of hematoporphyrin intraperitoneally, and then the tumors were exposed to light through a Lucite rod. Initial experiments were through intact skin, but in later experiments the authors placed the Lucite rod against the tumor through a skin incision. In all cases there was substantial destruction of the tumor; the only viable cells remaining were in a thin rim on the side furthest from the point of light application. At the time of treatment the tumors measured 1.5 to 2.0 cm in diameter. In 1978, Signorelli, et al., 8 reported on the photoradiation therapy (PRT) of human glioma cells in tissue culture. Cells from glioblastoma multiforme tumors were incubated in 10-5 M hematoporphyrin derivative (HPD) and then exposed to light from W neon light bulbs. Microscopic examination after treatment showed degenerative changes when compared to control cells. The cells tended to lose their cytoplasmic processes, showed large nuclear and cytoplasmic vacuoles, and lost their gliofibrillary structure. The quantitative aspects of treatment were not investigated. Sery 7 reported on the killing of retinoblastoma cells with HPD and light. The light source was a 15-W coolwhite fluorescent bulb giving an irradiance of 6.0 #W/ sq mm. He was also able to show that by increasing the plasma concentration the cytotoxic effect was markedly reduced, and heat-inactivated serum also inhibited the photodynamic effect. This result was thought to be due to binding of the porphyrin to hemopexin. Several groups have tried to treat patients with brain tumors using PRT and HPD. Perria, et al., 6 used a helium-neon laser that delivered 25 mw of power as a light source. Laws, et al, 5 and Forbes, et al., 3 used argon ion dye lasers as their light sources. All these groups reported evidence of tumor destruction in some patients, but no significant improvement in outcome was mentioned. We have been interested in applying PRT to brain tumors. The animal tumor model we have used is the rat 9L glioma model. This cell line is chemically produced by the administration of methylnitrosourea to pregnant rats and it has been widely used in braintumor studies. Previous studies with this model have used hematoporphyrin rather than HPD, and have used fluorescent light as the radiation source. The use of a J. Neurosurg. / Volume 64/May,

2 D. Hamilton, et al. laser has allowed us to accurately quantitate the light dose and has enabled us to assess factors such as wavelength, extracellular HPD concentration, and incident light intensity to determine the effectiveness of PRT. Glioma Cells Materials and Methods The 9L glioma cells* were grown as a subconfluent monolayer, then trypsinized for 10 minutes at 37"C. The trypsinized cell suspension was placed in a 15-ml centrifuge tube, and 5% fetal calf serum (FCS) in modified Eagle's medium (MEM) was added to fill the tube to the 15-ml level. The cells were counted and diluted further with 5% FCS in MEM to a final concentration of 4 x 104 cells in 2 ml. A 2-ml quantity of this suspension was then placed in every second well of a 4 x 6-compartment tissue culture tray. Alternate wells were used so that there would be no exposure of light from one well to another. Hematoporphyrin Derivative We used a commercial preparation of HPD known as Photofrin It which comes in a concentration of 5 mg/ml. This was diluted with 5% FCS in MEM to give the final concentration of drug. The cells were incubated for 24 hours in plain culture medium, which was then changed for medium containing HPD. The cells were treated 24 hours later. Laser Light The light source was a Coherent krypton ion laser or a Coherent Innova 20 argon ion laser pumping a rhodamine 6G dye laser.$ The output of the laser was passed through a 50% beam splitter where half of the beam went to the target and the other half went to a Coherent 210 Thermopile laser power meterw (Fig. 1). The target beam was expanded by means of a triplet lens, and the resulting beam was collimated using three adjustable shutters. The trays were mounted on a table fitted with a tube through which light could pass, thus isolating the treatment well from adjacent wells. In addition, an optical shield was placed over all wells not being exposed. The light passed through a hole in this shield of the exact diameter of the well and passed out through the floor of the tray and the hole in the table. The incident light was also measured with a power meter before and after each exposure. The first power meter measured the stability of the laser during the exposure, while a second was used to calibrate incident * The 9L glioma cells used in this study were obtained from the Brain Tumor Research Institute, University of California, San Francisco, California. t Hematoporphyrin derivative (Photofrin I) obtained from Photofrin Medical, Inc., Cheektowaga, New York. :~ Coherent ion lasers manufactured by Coherent, Inc., Palo Alto, California. w Coherent power meter manufactured by Coherent, Inc., Palo Alto, California. beam splitter ~~C illimjat or,/, 1 alignment mirror / protected ---I / culture wel~ 1 P / beam expander mounting tube ow.,..,., o,,,c., /L. ooo.ooo exposed culture well FIG. 1. Diagram of the experimental setup for administra- tion of laser light in vitro. Light from the laser passes through a lens system that expands the beam, which is then collimated and aligned so as to pass through the tissue culture well. Wells adjacent to the one being treated are protected from light exposure. A power meter monitors the output of the laser throughout the exposure to be sure that it is stable. A second power meter is used to measure the intensity of the incident beam before and after each exposure. light intensity before and after each well was exposed. The duration of the exposure was timed using a digitally controlled optical shutter. The wavelength of the incident light was measured with a Spex monochromator, Model 1870, U and compared to the settings of a calibrated triple-plate birefringent intracavity filter mounted in the Coherent 599 dye laser. The output mode of the laser was periodically checked by inspecting the image of the output beam by means of a concave mirror. The mode was kept at TM00 at all times. The expanded beam was truncated so that only the inside 30% of the beam was used. This ensured that there was a constant distribution of output power over the whole radius of the truncated beam. The image of the beam incident to the wells was checked for interference patterns that could arise from optical misalignments. Initially, this presented problems and caused alternating rings of viable and dead cells in the treatment wells. Since the output power of the dye laser is greater than 1 W at any wavelength used in this experiment, we could lose power in many places and still deliver a reliable monochromatic source of incident radiation. Also, since the output band width of the laser is less than 5 nm, this experimental configuration is a true source of monochromatic light that does not present the wavelength dosimetry problems of filtered white-light sources. The absorption spectrum of HPD in serum has five major peaks in the visible region, and this was used to select the treatment wavelengths. The argon ion and krypton ion lasers enabled us to examine the dose-, intensity-, and wavelength-dependence of the effect of PRT on 9L glioma cells. The wavelengths chosen were 647, 630, 590, 573, and 514 nm. The 647-nm output was delivered from a krypton ion laser with red mirror I[ Spex monochromator made by Spex Industries, Inc., Metuchen, New Jersey. or 776 J. Neurosurg. / Volume 64/May, 1986

3 Photoradiation of rat 9L gliosarcoma ~5o ~o g 1 I O O < 0.50 i i i i 0 i i 200 3go Wavelength (nm) FIG. 2. Absorption spectrum of hematoporphyrin derivative in saline. Tracing B is measured at greater sensitivity to show the spectrum over the visual range of wavelength. The shaded area shows the region of the spectrum where the effect of wavelength was studied. The arrows indicate those wavelengths studied, with three (514, 573, and 630 n m) corresponding to peaks in the spectrum and two (590 and 647 nm) corresponding to troughs. optics and an intracavity prism. The 630-, 590-, and 573-nm outputs were supplied by an argon ion laser pumping a rhodamine 6G dye laser. The 514-nm output was from the argon ion laser alone using green optics and an intracavity prism. The incident intensities used were 10, 30, 40, and 100 mw with doses ranging from 0.5 to 10 joules. Six wells were treated for each intensity, wavelength, and dose. The temperature of wells receiving the highest intensity and dose was measured with a copper constantine thermocouple and was found not to rise more than I*C above those not receiving laser treatment. Control Cells Control cells were incubated in the same trays as the treated cells so that they were handled in the same way. Some received only HPD and others only light, each at the same concentration of HPD and intensities and doses of light as the treated cells. They were stained with trypan blue, and in all cases there was no significant cell death. Estimation of Cell Death Immediately following treatment, the trays were returned to the incubator for 6 hours. The cells were then stained with trypan blue. It was not considered accurate to estimate the fraction of cell death or survival using the trypan blue technique, so the light dose and intensity were adjusted so as to produce 100% cell death. Effect of Wavelength Results The wavelength dependence of HPD phototherapy in vitro follows closely the HPD absorption spectrum. Figure 2 shows the HPD absorption spectrum in saline, with peaks occurring at 507, 540, 575, and 624 nm. Tracing B is measured at greater sensitivity than Tracing A. The shaded area shows the part of the spectrum over which the effect of wavelength was studied. Five differ- ~' 10 E o o c~ to 0.1 /.~\ "r 647 ~.~" ~;1590 "-~ I ~L4 H~3D concentration... 10mW/cm mW/cm mW/cm mW/cm 2 10 ~ molar s~o ' ~;o ' 6;0' o;o Wavelength (nm) FIG. 3. Graph showing the effect of wavelength on the dose of energy required to produce 100% cell kill. The error bars represent the highest dose delivered where there was not 100% cell death. More energy is required at wavelengths corresponding to troughs in the absorption spectrum (590 and 647 nm) than to peaks (514, 573, and 630 nm). Also, lower wavelengths and higher intensities are more efficient. HpD = hematoporphyrin derivative. ent wavelengths were selected, some corresponding to peaks on the spectrum (514, 573, and 630 nm) and others to troughs (590 and 647 nm). While 590 and 647 nm do not correspond to the lowest points in these troughs, they do represent wavelengths away from the peaks. Figure 3 shows the dose of light necessary to produce 100% cell kill at these five wavelengths at various intensities. The line error bars in all figures represent the highest dose delivered where there was not total cell death as determined by the trypan blue staining method. It can be seen that wavelengths of light corresponding to peaks in the spectrum are more effective in producing the photodynamic effect than are those in the troughs. For example, at a wavelength of 630 nm and an intensity of 100 mw/sq cm, only 0.8 joules/sq em is needed compared to 20 joules/sq cm at a wavelength of 647 nm. These results also show that a higher intensity is more effective than a lower one for any fixed total close of any wavelength. Also, those wavelengths that correspond to peaks in the absorption spectrum in the blue and green regions are more effective in killing the 9L glioma than are those in the red. Effect of HPD Concentration Figure 4 shows the effect of HPD concentration on PRT. As the HPD concentration increases so does the effectiveness of the treatment. These studies were all carried out with a 630-nm light setting as this is the wavelength used clinically. Again, less dose is required at higher intensities. These concentrations, ranging from 10-4 to 10-7 M, were chosen because they span the range of drug concentration found in brain tumors using this model (unpublished data). J. Neurosurg. / Volume 64/May,

4 D. Hamilton, et al. [] nm nm '~ nm [] nm DOsEIO / / (a/ cm 2) 1,0 10 DOSE (J/cm 2) 0.1_ 2 i2nm "4 10 S '7 Concentration of HpD (Molar) FIG. 4. Graph showing the effect of hematoporphyrin derivative (HpD) concentration on cell kill at two intensity levels and a wavelength of 630 nm. The higher the concentration, the more efficient is the photodynamic effect for any intensity of light i I I I I Intensity (mw/cm 2 ) FIG. 5. Graph showing the relationship between dose and intensity of light at various wavelengths. The total dose of energy only has meaning when the intensity is known, so the dose rate is important. This study also confirms that absorption peaks of lower wavelengths are more efficient than higher ones, as demonstrated in Fig. 3. Effect of Dose and Intensity of Light Figure 5 demonstrates the dose of energy required at various intensities and wavelengths to produce 100% cell kill. It can be seen that the total dose required at an intensity of 10 mw is five times that required at 100 mw. This graph also confirms that treatment wavelengths in the green region of the spectrum are more photocytotoxic than in the red. Discussion This study confirms that PRT destroys 9L glioma cells in vitro. Unlike the previous study on this cell line by Diamond, et al., 2 in our study we have isolated and quantified most of the parameters involved. The most efficient wavelengths for PRT are those that correspond to peaks in the absorption spectrum of HPD with the shorter wavelengths; that is, in the blue and green regions of the spectrum. This finding is in agreement with that of Kinsey, et al., 4 who also showed that green light is a very efficient wavelength to use in HPD PRT in tissue culture. Unfortunately, these lower wavelengths do not penetrate tissues as readily as those toward the red region of the spectrum, a factor of great importance when treating solid tumors. This has been well demonstrated in a study by Svaasand and Ellingsen 9 on the penetration of light into the brain as a function of wavelength. At 488 nm, light penetrated from 0.4 to 0.7 mm into the brain, whereas at 710 nm it traveled from 1.7 to 2.5 mm. At this distance, the intensity has fallen to 1/e or 37% of original value. Thus, in PRT the peak in the red part of the spectrum of HPD has to be used because of its ability to penetrate tissue, but this is at the expense of having less efficient killing of the malignant cells. For most efficient therapy using longer wavelengths, it is important that the laser be tuned so that its output is as close as possible to the absorption peak. Not all red light is equal, and the output of the dye laser should be checked regularly with a spectrometer to give optimal results. It can be seen that at 100 mw/sq cm and 630 nm it takes a dose of 0.8 joules/sq cm to give I00% cell kill compared to 20 joules/sq cm at 647 nm, a difference of only 17 nm in wavelength. Concentration studies show that the higher the concentration of HPD the more effective the treatment for any particular dose of light. At l0-6 M HPD, large doses of radiation are required to obtain 100% cell kill (8 joules at 100 mw/sq cm and 50 joules at 22 row/ sq cm). At l0-7 M HPD, we could not obtain 100% cell death at less than 100 mw/sq cm. It has been shown that high concentrations of HPD (5 x 10-4 M) have an inhibitory effect on cell growth in tissue culture, even in the absence of light.~ This effect varies between cell lines tested. In a previous study (unpublished data), we found the tumor concentrations of HPD 24 hours following administration of 10 mg/kg HPD intraperitoneally to be about 6 #g HPD/gm of tissue. This corresponds to a concentration of 10-5 M, a value giving some relevancy to the in vitro results in our present study. The in vitro phototoxicity of light and HPD depends 778 J. Neurosurg. / Volume 64/May, 1986

5 Photoradiation of rat 9L gliosarcoma on the dose rate. This has important implications when treating solid tumors since the intensity of light falls off rapidly with distance from the source, so that it is difficult to maintain an optimal dose rate remote from the light source. When this is considered along with the poor penetration of light through the brain, it becomes obvious that the main limitation in the application of PRT to brain tumors is going to be in delivery of light rather than drug uptake. References 1. Berns MW, Dahlman A, Johnson FM, et al: In vitro cellular effects of hematoporphyrin derivative. Cancer Res 42: , Diamond I, Granelli SG, McDonagh AF, et al" Photodynamic therapy of malignant tumours. Lancet 2: , Forbes I J, Cowled PA, Leons ASY, et al: Phototherapy of human tumors using haematoporphyrin derivative. Med J Aust 2: , Kinsey JH, Cortese DA, Moses HL, et al" Photodynamic effect of hematoporphyrin derivative as a function of optical spectrum and incident energy density. Cancer Res 41: , Laws ER Jr, Cortese DA, Kinsey JH, et al: Photoradiation therapy in the treatment of malignant brain tumors: a Phase I (feasibility) study. Neurosurgery 9: , Perria C, Capuzzo T, Cavagnaro G, et al: First attempts at the photodynamic treatment of human gliomas. J Neurosurg Sci 24: , Sery TW: Photodynamic killing of retinoblastoma cells with hematoporphyrin and light. Cancer Res 39:96-100, Signorelli CD, Ammirati M, Tajana G: Photochemotherapy of human glioma cells in culture by hematoporphyrin and visible light (preliminary experiment). Acta Neural (Napnli) 33: , Svaasand LO, Ellingsen R: Optical properties of human brain. Photochem Photobio138: , 1983 Manuscript received March 8, Accepted in final form October 23, This work was supported by grants from the Medical Research Council of Canada and Medical Services Inc. Foundation of Alberta. Address reprint requests to: John D. S. McKean, M.B.Ch.B., F.R.C.S.(C), Clinical Sciences Building, The University of Alberta, Edmonton, Alberta T6G 2G3, Canada. J. Neurosurg. / Volume 64/May,