Will J. Kort

Selective Accumulation of Endogenously Produced Porphyrins in a Liver Metastasis Model in Rats

The possibility of using the porphyrin precursor 5-aminolevulinic acid to cause selective porphyrin accumulation in tumors was examined. Syngeneic colon carcinomas CC531 were implanted in the livers of Wag/Rij rats. Groups of three to six animals each were given 2 mg/mL of 5-aminolevulinic acid in drinking water from the 8th, 14th, or 17th day after tumor implantation. Two other groups received either 2.5 or 5 mg/kg of Photofrin II (Photomedica Inc., Raritan, NJ) intravenously on day 17. On day 19 the livers were removed and porphyrin concentrations were measured in normal livers and tumors by solvent extraction and high-performance liquid chromatography. Protoporphyrin accumulated progressively in tumors with increasing duration of 5-aminolevulinic acid administration (P = 0.0001), whereas no increase was found in normal livers. After 11 days of 5-aminolevulinic acid administration the porphyrin concentration ratio between tumors and livers was 4:1. In contrast, after Photofrin II administration the concentration was higher in normal livers than in tumors (1:3 ratio, tumor to liver). Enzyme measurements showed a threefold decrease in ferrochelatase activity in tumors compared with livers (P less than 0.001). In conclusion, oral administration of 5-aminolevulinic acid results in progressive accumulation of protoporphyrin in a transplantable colon carcinoma without accumulation in the surrounding liver tissue. This selective accumulation of porphyrins appears to be caused by a relative ferrochelatase deficiency in malignant tissue. 5-Aminolevulinic acid administration may be a suitable approach to photosensitizing liver tumors for photodynamic therapy or to early detection of tumors by fluorescence in ultraviolet light.

Current Status of Photodynamic Therapy in Oncology

SummaryPhotodynamic therapy (PDT) is a cancer treatment based on the accumulation in malignant tissue of a photosensitiser with low systemic toxicity. Subsequent illumination induces a type II photochemical reaction with singlet oxygen production that results in destruction of biomolecules and subcellular organelles.The first full clinical report of PDT dates from 1976. Haematoporphyrin derivative, a complex mixture of porphyrins, was initially used as a photosensitiser. An enriched fraction (porfimer sodium) is now the most commonly used clinical agent. After systemic administration porphyrins bind to albumin and lipoproteins. Accumulation occurs mainly in tumours and organs of the reticuloendothelial system. The light of an argon-dye laser can be tuned to the appropriate wavelength and delivered either superficially, interstitially or intraluminally. Light distribution can be assessed by using a radiation transport model and tissue optical properties, or direct measurement with light detectors.The effects of PDT depend in a complex way on: characteristics, tissue concentration and localisation of the photosensitiser; the target tissue optical properties and oxygenation; activation wavelength, power density and treatment regimen. Future research is directed towards: better photosensitisers (i.e. phthalocyanines, chlorins or protoporphyrin IX endogenously produced from 5-aminolevulinic acid); improved light generation and delivery; and combination with hyperthermia, chemotherapy, radiotherapy or surgery. Adjuvant intraoperative PDT is a promising approach to destroying residual tumour after surgery.

Interstitial Nd:YAG laser coagulation with a cylindrical diffusing fiber tip in experimental liver metastases

Interstitial laser coagulation as a means of destructing hepatic metastases was investigated. Colon carcinoma CC531 was implanted in the liver of 42 Wag/Rij rats; 20 days later, tumors (5.5±0.2 mm) were exposed to 1,064 nm Nd:YAG laser light at 4 W/cm and either 600, 1,200, 2,400, 3,400, or 4,800 J/cm from a 0.5 cm Helioseal® coated cylindrical diffuser. Temperature and fluence rate were measured at the tumor boundary. Lesions were studied on day 2 and 36 posttreatment by light microscopy. Tumor proliferative activity was assessed by bromodeoxyuridine incorporation. Liver damage and function were determined by serum liver enzymes and antipyrine clearance. Fluence rate increased during laser treatment up to 170%; mean temperature increased logarithmically up to 69.7°C. Short‐term light microscopy showed coagulation necrosis of 7–11 mm without charring. Lesion size and liver enzymes increased logarithmically with laser energy applied. No deterioration in liver function was found. At 4,800 J/cm complete tumor remission occurred in three of four animals. This study shows the ability of interstitial laser coagulation to produce selective destruction of colonic tumor within the liver.

Effect of chronic light-dark shift stress on the immune response of the rat.

During a period of 35 weeks the night-day pattern of inbred Brown Norway female rats was changed weekly by alternating the light-dark (L-D) rhythm. After a period of 2 months, in a number of the animals, the cellular immune response was measured by means of Concanavalin A stimulation of peripheral blood (Con A) and a Popliteal Lymph Node Assay (PLNA). Serum corticosterone, plasma free fatty acids and peripheral leucocytes were determined as well. Seven months thereafter the remaining animals were sacrificed after which adrenal gland weight and spleen weight were established. Additionally, blood glucose and corticosterone were measured (corticosterone in vitro activity as well as the serum level). Both Con A and PLNA showed a significantly decreased immune response in the L-D shift stress group. Adrenal cortical activity measured in vitro as well as in vivo did not show any significant changes, neither at 2 months nor at 9 months. Therefore, the observed immunosuppressive effect of chronic light-dark shift stress can not be explained by an increased adrenal cortical activity. Other possible explanations for the effect of the light-dark shift stress on the immune response are discussed.

Adjuvant intraoperative photodynamic therapy diminishes the rate of local recurrence in a rat mammary tumour model

The use of photodynamic therapy (PDT) as an adjunct to curative tumour resection was investigated in a tumour recurrence model, using rat mammary adenocarcinoma BN472. Tumours were inoculated subcutaneously in 60 animals and resected after 21 days of growth. Immediately after removal, the operation site was exposed to 320-450 nm light of 0.1 W cm-2 and 60 J cm-2 after photosensitisation with either Photofrin (5 mg kg-1 i.v. 48 h before illumination) or 5-aminolaevulinic acid (ALA) (2 mg ml-1 in drinking water for 9 days). Porphyrin concentrations were measured in tissue samples. After 28 days, animals treated with adjunctive PDT had a significantly longer tumour-free interval than controls (P < 0.01); median 25 days (Photofrin), 18 days (ALA), 14 days (controls). Moreover, in the PDT groups significantly fewer rats had lymph node metastasis. A prophyrin concentration ratio between tumour and mammary tissue of 2:1 was found after Photofrin and 4:1 after ALA. The results indicate that adjuvant intraoperative PDT may be a safe and effective method of destroying residual tumour, thereby preventing locoregional tumour recurrence.

Modulation of metastatic ability by inhibition of cholesterol synthesis

Synvinolin (MK-733), a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG-CoA reductase) depressingde novo synthesis of cholesterol, was given to BN472 tumor cells in culture medium, 2 days prior to i.v. injection of the cells into syngeneic rats. Another group of rats received cells cultured under the same conditions but without synvinolin. Two different types of culture medium were used, a ‘complete’ medium (Hybridoma) and a medium (RPMI 1640) to which 1 per cent of fetal calf serum (FCS) was added. Tumor cells cultured in the presence of synvinolin showed significantly lower cholesterol values than untreated cells. Tumor cells treated with synvinolin had a decreased ability to form metastatic nodules when compared with control cells. The results supply further evidence for the suggestion that cholesterol modulates the ability of mononuclear cells to eliminate tumor cells, although it cannot be excluded that alteration of cell growth plays an important role as well.

Effect of stress and dietary fatty acids on allograft survival in the rat.

To determine the immunosuppressive effect of stress and a diet rich on linoleic acid on vascularized organ graft survival in the rat, a series of allogeneic heart and kidney grafts were carried out. Restraint stress resulted in a significant prolongation of the survival time. Although 5 days of postoperative stress gave the best results, 3 h of stress given on the first postoperative day already gave a marked prolongation of the survival time of kidney allografts. The beneficial effect of stress on heart survival was abolished when prior adrenalectomy was carried out. A diet high on polyunsaturated fatty acids gave significant prolongation of the survival times of kidney-grafted rats. A diet hgh on saturated fatty acids did not result in a statistical significant prolongation of the survival time, although the difference with the polyunsaturated fatty acid group was very small and not statistically significant. It was concluded that stress and diets high in certain types of fatty acids depress the immune response in rats, possibly via a mechanism which has some common pathways. The adrenal glands could play an important role in this immune inhibition.

Interstitial photodynamic therapy in a rat liver metastasis model.

Photodynamic therapy (PDT) of hepatic tumours has been restricted owing to the preferential retention of photosensitizers in liver tissue. We therefore investigated interstitial tumour illumination as a means of selective PDT. A piece of colon carcinoma CC531 was implanted in the liver of Wag/Rij rats. Photofrin was administered (5 mg kg-1 i.v.) 2 days before laser illumination. Tumours with a mean (+/- s.e.) diameter of 5.7 +/- 0.1 mm (n = 106, 20 days after implantation) were illuminated with 625 nm light, at 200 mW cm-1 from a 0.5 cm cylindrical diffuser and either 100, 200, 400, 800 or 1600 J cm-1. Control groups received either laser illumination only, Photofrin only or diffuser insertion only. Short-term effects were studied on the second day after illumination by light microscopy and computer-assisted integration of the circumference of damaged areas. Long-term effects were studied on day 36. To determine the biochemistry of liver damage and function, serum ASAT and ALAT levels were measured on day 1 and 2, and antipyrine clearance on day 1. Tumour and surrounding liver necrosis increased with light dose delivered (P < 0.001). Best long-term results were obtained at 800 J cm-1 with complete tumour remission in 4 out of 6 animals. No deterioration in liver function was found. The results of this study show the ability of interstitial PDT to cause major destruction of tumour tissue in the liver combined with minimal liver damage.

Treatment of experimental liver metastases with a noncontact neodymium : YAG laser

The extent of tumor and liver damage after treatment with the neodymium:yttrium-aluminum-garnet (Nd:YAG) laser was investigated in a rat tumor model. Colon carcinoma CC531 was implanted in the liver and treated with 60 J (14 tumors), 120 J (15 tumors), or 180 J (12 tumors) at a power setting of 20 W. To assess the effects upon the tissues three animals were sacrificed immediately after treatment and 1, 2, 4, and 8 days later. Sections from the tumor sites were evaluated by light microscopy and the maximal depth and width of different zones of tissue damage were measured. Laser effects could be determined most accurately on Days 1 and 2 after treatment. Multiple linear regression analysis of the data indicated a linear relationship between laser energy and depth of tumor damage (P less than 0.01). The results of this study show the potential of the Nd:YAG laser to produce tumor coagulation necrosis with minimal liver necrosis.

Photodynamic Therapy for Gastrointestinal Tumors

Photodynamic therapy is based on the administration of a compound that is preferentially accumulated by a tumor and which causes tumor destruction after exposure to light of a specific wavelength. The photosensitizers most commonly used in treating tumors of the gastrointestinal tract are porphyrins--hematoporphyrin derivative and dihematoporphyrin ether. These compounds have been used with success to produce reduction in tumor size of esophageal, gastric, and colorectal cancers. In some instances long-lasting complete remission have been observed after photodynamic therapy. New developments include photosensitizers that react to light of a longer wavelength, which is able to penetrate tissue to a greater depth, the use of 5-aminolevulinic acid, which is preferentially converted to porphyrin in malignant cells, and combination of photodynamic therapy with thermic laser, radiotherapy, or chemotherapy.