Experimental laser phototherapy of the morris 7777 hepatoma in the rat

0270-9139/85/0502-0175$02 OO/O

Copyright b 1985 by the American Association f o r the Study of Liver Dlseases HEPATOLOGY Vol. 5 , No. 2. pp. 175-180, 1985

Printed in U.S.A.

Experimental Laser Phototherapy of the Morris 7777 Hepatoma in the Rat

STEPHEN H O L T , ~ JOHN TULIP, DOUGLAS HAMILTON, JUDITH CUMMINS, ANTHONY FIELDS AND CATHERINE DICK

Department of Medicine and Department of Electrical Engineering, the University of Alberta, Edmonton, Alberta, Canada

The tumoricidal effect of the activation of hematoporphyrin derivative (HpD), by an argon-ion- dye-laser (wavelength 630 nm), was investigated in the Buffalo rat bearing subcutaneous implants of the Morris 7777 hepatoma. Tumor growth was monitored by measuring the tumor volume with constant force callipers. In control animals and those that were pretreated with HpD alone (10 or 20 mg per kg by i.p. injection) or laser light alone (2,000 J at 100 mW), a predictable exponential growth pattern of the cancer was observed. Animals were pretreated with HpD (10 mg per kg by i.p. injection) 48 hr prior to the fiberoptic, intratumor delivery of laser radiation (2,000 J at 100 mW), when the tumor had reached a volume greater than 1.5 cm3. Forty-eight hours after combined laser and HpD treatment, the hepatoma underwent coagulation necrosis, and the tumor volume rapidly increased from a mean value of 1.8 f 0.7 cm3 S.D. to a value of 6.8 f 1.5 cm3 S.D., compared with a mean of 3.7 f 0.7 cms S.D. in animals who had not received laser phototherapy. Rats treated with HpD and laser light survived longer (mean survival time 48 f 12 days S.D.) than did the other animals treated with the laser alone or HpD alone (mean survival time 31 f 16.5 S.D.). Tissue biodistribution studies with tritiated hematoporphyrin derivative, given with doses of 10 and 20 mg per kg of HpD, showed higher concentrations of the dye in the liver, kidneys and spleen than in the tumor at 48 hr after administration of the dye. The ratio of uptake of HpD between the Morris 7777 hepatoma and adjacent muscle was approximately 2:l in both dose ranges of the dye, implying that HpD does not localize in malignant tissue in a preferential manner. Laser phototherapy with HpD significantly interrupts the growth of the highly malignant Morris 7777 hepatoma and prolongs the survival time of Buffalo rats bearing this neoplasm.

The photodynamic effect of hematoporphyrin derivative (HpD) on malignant cells has been applied to the diagnosis (1) and treatment of cancer (2 ,3) . The exposure of mouse or rat tumors that contain HpD, to light of appropriate wavelength (630 nm) results in death of malignant cells. This photoradiation therapy has been curative in a number of tumor systems including cancer of the breast, prostate, colon, skin, endometrium, bone and in angiosarcoma (4, 5).

Earlier studies of the treatment of cancer by photoradiation have used a xenon lamp as a source of light for

Received January 17, 1984; accepted October 30, 1984. t Present address: Division of Gastroenterology, Cleveland Metro-

politan General Hospital, 3395 Scranton Road, Cleveland, Ohio 44109. This work was supported in part by a grant from the W. W. Cross

Cancer Institute and the Summa Medical Corporation of New Mexico. This paper has also been presented in part a t the Annual Meeting

of the Royal College of Physicians and Surgeons of Canada, September, 1983.

Address reprint requests to: Stephen Holt, M.D., Ch.B. M.R.C.P.(U.K.), F.R.C.P.(c), F.A.C.P., Division of Gastroenterology, Cleveland Metropolitan General Hospital. 3395 Scranton Road, Cleve- land, Ohio 44109.

HpD activation (5). More recently, light has been delivered by a laser (6). A laser, combined with a fiberoptic delivery system, is a more efficient and convenient light source because the laser wavelength may be selected to fall in the optimal range for HpD activation and the fiber may be used to deliver light percutaneously.

HpD may accumulate preferentially in neoplastic cells ( l? 7 ) . However, the generalization that HpD reaches a concentration in malignant tissue to a higher degree than in most normal tissue (1,7,8) has not been substantiated (9). A knowledge of the biodistribution of HpD is an important part of any phototherapy study because the therapeutic ratio in laser phototherapy depends upon the concentration of HpD in normal tissue compared with neoplastic tissue.

To further investigate the use of phototherapy for the treatment of cancer, HpD was administered to Buffalo rats bearing subcutaneous implants of the Morris 7777 hepat,oma, and these tumors were activated by light from an argon-ion-dye-laser. Tumor growth was observed, and in separate experiments the biodistribution of HpD, in tumor bearing rats, was determined.

K'l' AL. HEPATOLOGY

MATERIALS AND METHODS TUMOR MODEL

The poorly differentiated and rapidly growing Morris 7777 hepatoma was selected from tissue culture for subcutaneous implantaLion in Buffalo rats (Simonson Labs, Gilroy, Calif.; mean weight 251 k 5 S.E. gm). Throughout all studies, each animal was allowed free access to Purina Laboratory Chow and water. The Morris 7777 hepatoma is a nonmetastasizing transplantable tumor that arose initially in the Buffalo rat after treatment of the animal with the alkylating agent, 2-N-fluorenyl phthalamic acid. The untreated hepatoma grows rapidly, and it reaches a size of about 10% of body weight of the animal within 4 to 5 weeks (10).

Initial studies, using subcutaneous trocar implants of the cancer in both flanks of six rats, revealed consistent but eccentric tumor growth and death of each animal within 4 to 5 weeks. To produce a tumor model with predictable growth characteristics, a skin incision was made on each flank of five Buffalo rats and a piece of tumor (approximately 2 mm in diameter), derived from an already established subcutaneous growth in a separate animal, was transplanted. The incision was then closed with fine silk sutures. Tumor growth was monitored by daily measurements of the tumor volume with constant force callipers (10 g force), used in three orthogonal planes. The double-fold skin thickness was subtracted from each measurement, and the corrected diameters were used to calculate an ellipsoidal tumor volume. Tu- mor growth occurred in a reproducible and linear pattern (Figure 1). After reaching a volume of 1 cm, the hepatoma grew in an exponential manner in all animals. The growth of the tumors was found to be exponential between volumes of 1 and 6.5 cm3 with a mean doubling time of 4.1 days. The mean doubling time was derived from the linear regression of the logarithm of the tumor volumes against the number of days after reaching a volume of 1 cm3. Throughout this and all subsequent studies, direct implantation of tumors was undertaken into both flanks of each animal, and the animals were anesthesized with halothane during experimental han- dling.

4.0, 4%

2.0+,

0 5 10 15 20 25 0 5 10 15 20 25

Days After Inoculation Days After Inoculation

L e f t F l a n k Right Flank

FIG. 1. Control group mean growth curves. The mean loglo tumor volume (hepatoma) in the right and left flanks of the Buffalo rat plotted against time indicating exponential growth. Data are mean f S.D. DT, doubling time.

BIODISTRIBUTION OF 'H-HPD The biodistribution of tritiated hematoporphyrin de-

rivative ('H-HpD, New England Nuclear, Boston, Mass.) was determined in two groups of six Buffalo rats bearing the Morris 7777 hepatoma in their left and right flanks, using previously described and validated methods (9). The comparability of 3H-HpD and cold HpD (Photofrin) was assessed by high-pressure liquid chromatography, thin-layer chromatography and measurement of absorption spectra of these two compounds (9).

In the first group of six tumor-bearing rats, a 10 mg per kg dose of 3H-HpD and in the second group of six rats, a 20 mg per kg dose of 3H-HpD were administered by i.p. injection when the neoplasms had reached an approximate volume of 2 cm. Animals in both experimental groups were sacrificed 48 hr following 3H-HpD administration, and whole blood (0.5 to 0.7 ml) and tissue samples (30 to 70 mg) of tumor, liver, kidney, brain, spleen, lung and muscle were collected after dissection of each animal. The wet weight of each sample was recorded, and tissues were solubilized in Protosol (New England Nuclear) for 18 hr a t 55°C. Samples were then bleached with stabilized hydrogen peroxide and neutral- ized with glacial acetic acid. After the addition of scintillation cocktail, the treated tissue samples were dark- adapted for a further 18 hr. Counting was undertaken using a Beckman Model L.S. 9800 liquid scintillation counter with standards to determine quench. Corrections were made for efficiency and quench. The resulting cpm for each sample were converted to dpm and then trans- formed to weights of radioactive HpD. Data were ex- pressed as the mean weight of HpD (pg) per gram of tissue for six specimens k one standard deviation. Sta- tistical comparisons were made using the Student's t test.

PHOTOTHERAPY Five groups (Table 1) of Buffalo rats received a surgical

implantation of the Morris 7777 hepatoma in the manner as previously described. Each animal had a tumor in the left and right flank. Group 1 (Figure 1) received no treatment and formed a control group. Groups 2 to 5 formed the treatment groups. These tumors were treated when they had reached a volume of 1.5 cm3. Treatments consisted of the intratumor delivery of laser light alone (2,000 J at 100 mW) (Group 2), HpD given by i.p. injection in doses of 10 mg per kg (Group 3) or 20 mg

TABLE 1. FIVE GROUPS OF RATS USED TO STUDY LASER PHOTOTHERAPY OF THE MORRIS 7177 HEPATOMA"

Group Experimental conditions No. of animals

1 No treatment 5 2 Laser light alone (2,000 J at 100 5

3 HPD (10 mg/kg) 5 4 HPD (20 mg/kg) 5 5 HpD (10 mg/kg) with laser light 8

mW)

(2,000 J at 100 mW) Each animal had two tumors, one in the left flank and one in the

right flank.

Vol. 5, No. 2, 1985 LASER PHOTOTHERAPY OF HEPATOMA 177

per kg (Group 4) alone and intratumor delivery of laser light (1,000 to 2,000 J at 100 mW) 48 hr after the i.p. administration of HpD (10 mg per kg) (Group 5). Al- though sham treatments were not performed in a controlled manner, no major effect on the growth had been noted by placement of the probe during preliminary trials. Tumor growth was monitored by measuring the tumor volume with callipers, and the length of survival of each animal was noted. Autopsy was performed on at least two animals from each group following their death, and adjacent muscle and tumor tissue was removed for light and electron microscopy from animals before and after laser phototherapy (Group 5).

The delivery of light to the tumors was performed using a 600 pm D, step index fiber (quartz silica) that was implanted interstitially along the longest axis of the tumor. Although this technique provided the most uniform transillumination of the neoplasms, a rapid de- crease of transillumination intensity occurred at the tumor surface following the initiation of the laser treatment. This reduction of transillumination was a result of the baking of tissue and blood onto the fiber tip with consequent light absorption and heating.

To overcome this problem, about 1 cm of the distal end of the fiber was treated with acrylic cement. The cement dried onto the quartz core of the fiber to produce a mat surface. The refractive index of the acrylic cement closely matched that of the quartz core so that light readily passed from the core into the coating where it was scattered by the rough outer surface. Hence, the light leaked out of the fiber along the length of the treated tip resulting in a cylindrical radiator, not unlike a miniature fluorescent lamp.

Before laser light was administered, the skin overlying the tumor was shaved and the animal was immobilized on a small metal frame. A 21-gauge needle was inserted into the longest axis of the tumor and 500 pl of normal saline were injected into the center of the tumor. The acrylic coated fiber, through which light was transmitted from the argon-ion-dye laser, was inserted into the needle and the needle was then withdrawn to expose the fiber tip. The fiber tip was then located in the approximate middle of the neoplasm, and laser light was administered at a rate of approximately 1 J per 10 sec. During light delivery, the intratumor and intraperitoneal temperature of each animal were monitored simultaneously with a 30-gauge copper thermocouple.

RESULTS

BIODISTRIBUTION OF :3H-H~D

No significant variation was detected between cold HpD and 3H-HpD by thin-layer chromatography, high- pressure liquid chromatography or measurement of the absorption spectra of these two compounds. The resulting chromatographic pattern of 'H-HpD appeared to be the same as that of cold HpD (Photofrin, Photofrin Medical Inc., Cheektowaga, NY), and radioactivity was assayed to be present only in the three chromatographic bands (10, 4, 3) that are representative of HpD. These

data confirm earlier findings of Gomer and Dougherty (1979).

The results of blood concentrations and tissue distribution of 3H-HpD (10 and 20 mg per kg), 48 hr after its i.p. administration to the two groups of Buffalo rats, are shown in Figure 2, a and b. Tissue distributions of 'H-HpD were similar at both doses of 3H-HpD. Of the eight tissues that were assayed in each group of animals that received either 10 or 20 mg per kg, the liver, kidney and spleen contained significantly higher concentrations of porphyrin (at 10 mg per kg of HpD, mean concentrations-liver = 43.2 f 5.4 S.D. pg per gm, kidney = 52.4 ? 6.8 S.D. pg per gm, spleen = 30.6 f 8.1 S.D. pg per gm; at 20 mg per kg of HpD, mean concentration-liver = 70.3 f 11.1 S.D. pg per gm, kidney = 91.8 & 13.5 pg per kg, spleen = 50.7 & 14.7 S.D. pg per kg) than the tumor at 48 hr (at a dose of 10 mg per kg of HpD, mean concentrations-right tumor = 5.0 k 0.5 S.D. pg per gm, left tumor = 4.0 f 1 S.D. pg per gm; at a dose of 20 mg per kg of HpD, mean concentrations-right tumor = 9.2 f 2.5 S.D. pg per gm, left tumor = 8.9 f 2.6 S.D. pg per gm) (p < 0.01 for concentrations in each listed organ compared with tumor concentrations, Student's t test). There appeared to be as much of 3H-HpD in the lung and blood as in both tumors for each dose level (Figure 2, a and b). The tumor to muscle ratio of HpD was approximately 2:1, and less of the porphyrin was present in the brain than in the tumor (Figure 2, a and b).

LASER PHOTOTHERAPY The results of treatment in 4 of the 5 groups of rats

are shown in Figure 3. Neither HpD alone (at 10 mg per kg), or laser light alone (2,000 J at 100 mW) caused any measurable effect on normal tumor growth. Group 4 received HpD alone in a dose of 20 mg per kg, and tumor growth was unaffected. After the combined administration of HpD (10 mg per kg) by i.p. injection and laser light, there was a rapid increase in tumor size which was generally noticeable, on inspection of the animal, within 6 hr of light delivery (Figure 3). The hard, nodular nature of the subcutaneous hepatoma changed to a soft mass on palpation, and the tumor often became a cystic swelling. Forty-eight hours after combined laser and HpD treatment, the hepatoma underwent coagulation necrosis, and the tumor volume rapidly increased from a mean value of 1.8 f 0.7 cm' S.D. to a value of 5.8 f 1.5 cm3 S.D., compared with a mean of 3.7 k 0.7 cm3 S.D. in animals who had not received laser phototherapy. In one animal, that had received laser phototherapy, tumor tissue leaked when the skin over the treated tumor was punctured with a needle. In 2 of the 8 rats treated with laser phototherapy, some ulceration of skin over the tumor was noted. The rats behaved sluggishly for approximately 2 days after treatment but then returned to apparently normal activity. In 6 of 8 of the animals that received laser phototherapy with HpD, the tumor ulcerated and sloughed away. This striking tumoricidal effect with liquefaction of tissue made subsequent measurements of tumor volume difficult but within 2 to 5 days of treatment, tumor growth appeared to recover. The mean

178 HOLT ET AL.

10 rng/ kg HPD48 Hours Post I.P. Injection

90 -

80 -

70 -

60 -

\ 50 0) a

-

100

80

70

80

E, . 5 0 m

4E

3c

2c Data are means f S.D.

1c

HEPATOLOGY

20 mg/ kg HPD48 Hours Post I.P. Injection

L

Data are means + S.D.

hnizln FIG. 2. The concentration of HpD in various tissues 48 hr following the i.p. administration of 3H-HpD in doses of 10 mg per kg of HpD (a)

and 20 mg per kg of HpD (b). Data are mean * S.D.

3.5-

i

a P 0 I 2 3.0-

0

9

- = HPD ( I O ~ l K q ) A L I W T ZOOOJ - = CONTROL HPD lIOrng/Kg) ALONE

-4 = LIGHT 12000JlALONE

2 5 1 . ( f , , , , , , , i , , , , , . , . . I I

- I - S - J - * J - ~ . I D I 2 3 ~ 1 6 7 0 9 1 0 1 1 1 2 1 3 n

DAYS AFTER REACHING lcm3

FIG. 3. Change in tumor volume with time in four groups of rats receiving the following: no treatment (Group 1) or treated by laser light alone (Group 2); HpD (10 mg per kg) alone (Group 3), or HpD (10 mg per kg) and laser light (2,000 J) (Group 5). HpD combined with laser light resulted in an increase in the volume of the tumor between 6 and 48 hr posttreatment. Data are means; error bars are omitted for clarity.

survival time, after the implantation of the hepatoma, in the rats treated with light and HpD was significantly longer (48 f 12 S.D. days) than was the mean survival in all other groups of animals (31 2 16.5 S.D. days (p < 0.05).

Measurements of temperature in the tumor, within 0.1 cm of the surface of the fiberoptic, light-delivery cable, were uniformly less than 3°C above the simultaneously measured, i.p. temperature of the animal. This implied

that the resultant light pattern did not give rise to any "hot spots" at the doses of light used in these experiments, and thermal injury was not responsible for the observed tumoricidal effects of laser phototherapy with HpD. Autopsies, performed after death of the animals, showed advanced malignancy which was manifest most often by direct intraperitoneal invasion of the hepatoma through the muscles of the flank of the rat. Macroscopic examination of the abdominal and thoracic organs after death did not reveal major differences between each group of animals, and the pattern of tumor invasion appeared similar in all groups.

Light microscopy of sections of the biopsies of tumor tissue that had been removed before and after laser phototherapy revealed the development of extensive coagulation necrosis in the treated neoplasm with total cellular disruption that could be attributed to therapy. This finding far exceeded the small and infrequent areas of spontaneous necrosis that were evident by light microscopy in tumors that were not treated by phototherapy. However, there was some evidence of survival of islands of hepatoma cells within the tissue sections removed from neoplasms that had been treated with laser phototherapy and HpD. The finding of surviving hepatoma tissue matched the observation of recurrent tumor growth in the animals after treatment (Figure 3). Most necrosis was noted in the middle of the tumors a t areas of the tumor adjacent to entry of the fiberoptic light cable. Sections of hepatoma tissue, removed from tumors approximately 2.5 cm3, were examined after treatment

Vol. 5, No. 2, 1985 LASER PHOTOTHERAPY OF HEPATOMA 179

with HpD alone or laser light alone. They revealed only small and infrequent areas of necrosis that were detectable only by light microscopy. Electron microscopy of tissue sections after laser phototherapy showed disruption of cellular organelles with mitochondria1 rupture, nuclear disruption and extensive cross-linking of cell membranes. Such changes at light or electron microscopy were not seen in the other groups of animals that received HpD alone (10 or 20 mg per kg) or laser light alone.

DISCUSSION Laser phototherapy with HpD significantly interrupts

the growth of the highly malignant Morris 7777 hepatoma and improves survival time in the Buffalo rat bearing this tumor. The data from these studies confirm the findings of other investigators (2, 4, 10-12) where light activation of phototoxic dyes, such as HpD, has been found to have important cancer treatment properties.

Although tumors in animals exhibit marked fluorescence when treated with HpD, our data does not indicate selective localization of this porphyrin to a degree that has been initially supposed (4, 7). Other studies have tended to assume preferential concentration of HpD in malignant tissue because of enhanced tissue fluorescence in tumors that have been pretreated with the dye (7). However, Gomer and Dougherty (1979) have suggested that the apparent degree of brightness of fluorescence depends on both the depth of tissue penetration of light and transmission of the fluorescent wavelengths. Since these properties vary depending upon the degree of pig- mentation or blood content of tissues or organs, the degree of fluorescence cannot be related directly to the amount of HpD contained in the tissue. In addition, earlier attempts to quantitate the distribution of HpD in uivo (4, 7) have utilized fluorometric and spectrophoto- metric techniques, each of which may be inaccurate because of fluorescent quenching by tissue pigments.

The photooxidative effect of HpD and light appears to be mediated by the production of singlet oxygen that is formed by energy transfer from the porphyrin to endog- enous oxygen (13). Cell death is believed to result from this oxidation and cross-linking of membrane compo- nents with ensuing cell lysis (14). In this study, the histological findings at light and electron microscopy in the hepatoma treated with laser phototherapy illustrate the dramatic cytotoxic effect of the treatment modality and confirm earlier observations (13,14). The toxic effect of HpD phototherapy on neoplasms is not specific, since normal tissue containing HpD can be damaged during illumination. However, in the present study, light microscopy of muscle tissue adjacent to tumors treated with HpD did not reveal evidence of necrosis. Despite an apparent nonspecific localization of HpD, good therapeutic ratios between tumor response and skin response (phototoxic eruptions) have been reported in clinical trials (5, 14). However, severe and persistent phototoxic reactions to hematoporphyrin administration can be an- ticipated in a significant proportion of patients, thereby limiting the clinical use of phototherapy (15).

It would appear that more localized delivery of HpD

to tumors by topical application or by conjugation with a tumor-seeking compound such as monoclonal antibody or a lectin has potential to reduce toxicity to normal tissue. Normal tissue adjacent to a neoplasm could be severely damaged if the timing between HpD administration and light exposure is not optimal. In addition, the therapeutic ratio obtained during phototherapy depends to a large extent on the location of the tumor. The liver is an organ which may pose problems for photoradiation treatment because of its ability to concentrate HpD. The treatment of intrahepatic malignancy by HpD and light delivered percutaneously through a needle inserted into primary or secondary malignancies in liver, or the use of phototherapy as an adjunct to the treatment of hepatic neoplasia, are intriguing possibilities. How- ever, they may not be feasible without a selective delivery system for HpD or other photoactive dyes.

Our data does not support the notion that HpD locates preferentially in malignant tissue and is in agreement with the findings of Gomer and Dougherty (1979), where high concentrations of HpD were detectable in the liver, kidneys and spleen of tumor-bearing mice. Selective delivery of HpD to a neoplasm by a tumor-seeking compound such as a monclonal antibody (16) has obvious advantages since it may be possible to reduce the systemic toxicity of photoactivated dyes. The concept of photoimmunotherapy has been proposed by Mew et al. (1983) where HpD conjugated with a tumor-seeking monoclonal antibody appeared to have cancer treatment properties in a mouse tumor model. The theoretical advantages of photoimmunotherapy include a possible reduction in dose levels of HpD and reduction of concentration of the dye in adjacent tissues and skin, thereby reducing local and systemic toxicity.

The mechanism of localization of HpD in malignant tissue is unknown, and recent studies would suggest that there may be species difference in its biodistribution (9). It has been suggested that human tumors may accumulate more HpD and retain it for a longer time than murine tumors (5, 14). Since blood flow is most likely a major determinant of HpD localization, then necrosis of normal tissue adjacent to or within the hepatic neoplasm may be a limiting factor for the concentration of the dye. In addition, the uptake tumor-seeking conjugates of HpD with monoclonal antibodies directed against neoplasms, or other tumor-seeking compounds, would be limited by such necrosis. However, small areas of spontaneous necrosis that occurred in areas of the Morris 7777 hepatoma in the Buffalo rat did not appear to interfere with laser phototherapy in this experiment model. Hematopor- phyrin derivative seems to accumulate in a wide variety of neoplasms (9, 5, 14) but the effect of factors such as cell type and degree of differentiation of the tumor on HpD uptake have not been studied in detail. In our study, we were unable to observe effects on HpD uptake that could be attributed to the degree of differentiation of the Morris 7777 hepatoma.

Major factors to be considered in clinical application of phototherapy include efficient and selective light activation of HpD containing malignant tissue. The mod- ification of the tip of the quartz fiber that was used in

180 HOLT ET AL. HEPATOLOGY

the laser system in this experiment appears to possess some advantages for efficient light delivery. We have noted that if the treated length of the fiber in this system was made equal to the reflection length inside the fiber, then a uniform cylindrical radiation source resulted. This offered clear advantages, as an interstitial source, over a simple cleaved fiber. The power flux of 100 mW produced an intensity of approximately 30 W per cm2 at the tip of a 0.6 mm fiber, whereas the same power flux in a cylindrical radiator would be 0.5 W per cm2 at the surface. In consequence, local heating at the fiber tip was reduced considerably. A further and less obvious advantage of a cylindrical source over a point source is the relative geometric fall in light intensity as the light propagates away from the source. In the case of a point source, the source has spherical geometry so that the geometrical reduction of intensity follows the cubed power of the distance from the source. For a cylindrical radiator, this reduction in intensity follows the squared power of distance so that uniformity of transillumination is conse- quently improved. The mode of light delivery that was used in the present study would be suitable for light delivery to endoscopically accessible tumors or malignant lesions that are within reach of a percutaneous needle puncture. In the present study, only one period of activation of HpD by a laser was used. It may be possible to treat malignancy more effectively with repeated sessions of laser phototherapy. Recently, we have modified our light delivery system for multiple single fiber probes which will permit powerful illumination of a larger area of a neoplasm.

The demonstration of the lack of preferential uptake of HpD by neoplastic tissue in the present tumor model in comparison to some other organs has important im- plications for the application of phototherapy. The use of a controlled point source of light delivery by a laser could lead to less damage to adjacent tissue but the presence of significant amounts of HpD at other areas, such as adjacent tissue and the skin, are important determinants of toxicity. The rapid and striking tumoricidal effects of photoactivated HpD show great promise

for the treatment of hepatic and other tumors. However, this treatment modality for deep-seated tumors may become acceptable for clinical use only when a combi- nation of selective tumor delivery of HpD together with efficient light delivery can be achieved.

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Experimental laser phototherapy of the morris 7777 hepatoma in the rat