OXYGEN LIMITATION OF DIRECT TUMOR CELL KILL DURING PHOTODYNAMIC TREATMENT OF A MURINE TUMOR MODEL

The relationship between levels of in vivo accumulated photosensitizer (Photofrin II), photodynamic cell inactivation upon in vitro or in vivo illumination, and changing tumor oxygenation was studied in the radiation-induced fibrosarcoma (RIF) mouse tumor model. In vivo porphyrin uptake by tumor cells was assessed by using 14C-labeled photosensitizer, and found to be linear with injected photosensitizer dose over a range of 10 to 100 mg/kg. Cellular photosensitivity upon exposure in vitro to 630 nm light also varied linearly with in vivo accumulated photosensitizer levels in the range of 25 to 100 mg/kg injected Photofrin II, but was reduced at 10 mg/kg. Insignificant increases in direct photodynamic cell inactivation were observed following in vivo light exposure (135 J/cm2, 630 nm) with increasing cellular porphyrin levels. These data were inconsistent with expected results based on in vitro studies. Assessment of vascular occlusion and hypoxic cell fractions following photodynamic tumor treatment showed the development of significant tumor hypoxia, particularly at 50 and 100 mg/kg of Photofrin II, following very brief light exposures (1 min, 4.5 J/cm2). The mean hyupoxic cell fractions of 25 to 30% in these tumors corresponded closely with the surviving cell fractions found after tumor treatment in vivo, indicating that these hypoxic cells had been protected from PDT damage. Inoculation of tumor cells, isolated from tumors after porphyrin exposure, into porphyrin-free hosts, followed by in vivo external light treatment, resulted in tumor control in the absence of vascular tumor bed effects at high photosensitizer doses only.(ABSTRACT TRUNCATED AT 250 WORDS)     

TUMOR DESTRUCTION IN PHOTODYNAMIC THERAPY

Abstract The effects of photodynamic therapy (PDT) on the tumor microvasculature in the first few hours after treatment was studied at the light microscope (LM) and electron microscope (EM) levels in DBA/2Ha mice bearing SMT-F tumors. Animals received intraperitoneal injections of 10 mg kg⁻ of Photofrin II and 24 h later tumors were treated with 100 J cm⁻² of light (630 nm). Animals were sacrificed and their tumors removed at time 0, 30 min, 1, 2, 4, 8, 16 and 24 h after treatment. The results indicate that the effects of PDT are initially direct destruction of the microfibrils in the subendothelial zone of the tumor capillaries with subsequent tumor cell death secondary to hemorrhage and vascular collapse.     

Photodynamic Therapy of 9L Gliosarcoma with Liposome-Delivered Photofrin

Abstract— The effect of Photofrin encapsulated in a liposome delivery vehicle for photodynamic therapy (PDT) of the 9L gliosarcoma and normal rat brain was tested. We hypothesized that the liposome vehicle enhances therapeutic efficacy, possibly by increasing tumor tissue concentration of Photofrin. Male Fisher rats bearing a 9L gliosarcoma were treated 16 days after intracerebral tumor implantation with either Photofrin in dextrose (n = 5) or Photofrin in liposome (n = 6). Nontumor-bearing animals were treated with Photofrin delivered either in dextrose (n = 4) or liposome (n = 4) vehicle. Tissue concentrations of Photofrin delivered either in dextrose (n = 4) or liposome (n = 4) vehicle were measured in tumor, brain adjacent to tumor and in normal brain tissue. Photofrin was administered (intraperitoneally) at a dose of 12.5 mg/kg and PDT (17 J/cm₂ of 632 nm light at 100 mW/cm₂) was performed 24 h after Photofrin administration. Brains were removed 24 h after PDT and stained with hematoxylin and eosin for analysis of cellular damage. The PDT using Photofrin in the liposome vehicle caused significantly more damage to the tumor ( P < 0.001) than did PDT with Photofrin in dextrose. The PDT of tumor with Photofrin delivered in liposomes caused a 22% volume of cellular necrosis, while PDT of tumor with Photofrin delivered in dextrose caused only scattered cellular damage. Photofrin concentration in tumors was significantly higher ( P = 0.021) using liposome (33.8 ± 18.9 μg/g) compared to dextrose delivery (5.5 ± 1.5 μg/g). Normal brain was affected similarly in both groups, with only scattered cellular necrosis. Our data suggest that the liposome vehicle enhances the therapeutic efficacy of PDT treatment of 9L tumors.     

The effect of photodynamic therapy alone and in combination with misonidazole or X-rays for management of a retinoblastoma-like tumour

Abstract— The effect of photodynamic therapy alone and combined with misonidazole or X-rays has been investigated in an intraocular retinoblastoma-like tumor. Tumour control rate was achieved up to 33% and depended both on the light energy doses and the Photofrin II doses. Misonidazole injected prior to light irradiation did not enhance the photodynamic therapy response, although the misonidazole was uptaken by the tumor tissue. The combination of X-rays and photodynamic therapy demonstrated both in vivo and in vitro not more than a simple additive effect and there was no difference between X-rays given before or after the light irradiation.     

IMPROVED SURVIVAL FROM INTRACAVITARY PHOTODYNAMIC THERAPY OF RAT GLIOMA

The effectiveness of intratumoral photoradiation in photodynamic therapy (PDT) using a polyporphyrin photosensitizer was studied in the RT-2 rat glioma model. One week after intracerebral implantation of RT-2 cells, experimental rats received a single i.p. injection of 2 mg/kg of Photofrin. After administration of the photosensitizer (48 h), the tumors were partially resected and the exposed cavity was irradiated with 15 J of laser light at a wavelength of 630 nm. Further treatment with a large craniectomy significantly enhanced rat survival. Control rats which received no photosensitizer but were treated with surgery, alone or in combination with laser irradiation, succumbed from early tumor recurrence. Photodynamic therapy without decompressive surgery resulted in hemorrhagic infarction of residual tumor and adjacent brain with focal cerebral edema which resulted in cerebral herniation and early death. Our results indicate that photodynamic therapy is effective in treating residual brain tumor but at the expense of brain tissue surrounding the tumor. Unless relieved, intracranial pressure from photodynamic therapy-associated cerebral edema in this animal model resulted in shortened survival.     

EARLY BIOCHEMICAL RESPONSES TO PHOTODYNAMIC THERAPY MONITORED BY NMR SPECTROSCOPY

Abstract Studies directed at determining the biochemical events that lead to tumor cytotoxicity following photodynamic therapy, a promising new approach for treatment of neoplasia, have demonstrated that exposure of R3230AC mammary tumors to hematoporphyrin derivative or Photofrin II plus visible light caused marked impairment of mitochondrial enzymes functioning in oxidative phosphorylation and electron transport. ³¹P-NMR spectroscopy has now demonstrated that a rapid and striking decrease in NTP (ATP) levels, concomitant with a marked increase in P_;, occurs in tumors shortly after photodynamic therapy. These effects appear to be fluence related. Possible changes in tumor vascularity, as detected by ²H-NMR measurements of the uptake of D₂0, were not observed under the conditions studied. Taken together with our earlier results, we conclude that the reduction in tumor ATP levels in situ , probably via inhibition of mitochondrial function, is a direct and early response of neoplastic tissue to porphyrin-induced photosensitization.     

Changes in tumor interstitial pressure induced by photodynamic therapy.

This study has examined the changes in tumor interstitial pressure exhibited during and after photodynamic therapy (PDT). The kinetics of these changes are marked by an initial decrease, followed by a rapid rise in tumor interstitial pressure. We have also employed two inhibitory agents to evaluate the different components of the pressure curve. Specially designed pressure chambers were seeded with chondrosarcoma and implanted subcutaneously in rats. Animals were injected with 0-50 mg/kg Photofrin II (i.v.) 7 days post-implantation and tumors were exposed to 0-540 J/cm2 630 nm 24 h later. Interstitial pressure was monitored via a transducer connected to the implanted chamber. Additional groups of animals were injected with either indomethacin (an inhibitor of thromboxane synthesis) or Ketanserin (a serotonin antagonist) before light treatment. Porphyrin doses of 10 mg/kg and above (135 J/cm2), or light doses of 135 J/cm2 and above (25 mg/kg Photofrin II) were effective in modifying interstitial pressure. Porphyrin doses greater than 25 mg/kg, or light doses greater than 270 J/cm2 produced no further increases in interstitial pressure. Animals given indomethacin (10 mg/kg i.p.) exhibited the initial decrease in pressure during light treatment, but showed no increase past baseline levels. Animals given Ketanserin (10 mg/kg i.p.) demonstrated no decrease in pressure during PDT, but showed the same elevations in pressure as controls. This suggests that two independent mechanisms account for the different components of the pressure curve, and that serotonin release may occur during PDT.     

INTRATUMOR TEMPERATURE MEASUREMENTS DURING PHOTODYNAMIC THERAPY

Abstract Photodynamic therapy has been under investigation as a form of cancer treatment for a number of years. This procedure uses a light source of 630 nm to photoactivate the drug, Photofrin II. Researchers in the past have reported temperature increases during photodynamic therapy, by measuring surface temperature of the tumor or a single point temperature within the tumors. Three temperature points within the tumors have been measured in this study, to quantify the temperature distribution within the lesion. These temperatures were measured for photodynamic therapy treated mice and control mice receiving an exposure to the treatment light without the drug. The use of a filtered xenon arc lamp for the 630 nm light source produced larger temperature increases and thermal gradients within the tumors, than when an Argon dye laser was employed. This temperature increase is due in part to the broad wavelength output of this filtered lamp. When this thermal effect is present during PDT treatment, researchers have observed the development of shock proteins resulting in the induction of thermotolerance and resistance to subsequence hyperthermia treatments. Using the filtered arc lamp, mice receiving photodynamic therapy treatments displayed consistently higher temperature increases than control mice. The use of an argon dye laser, with sufficient air cooling of the tumor, can eliminate this thermal effect. It has been demonstrated that the use of filtered lamps produce thermal effects which cannot be eliminated, demonstrating that lasers should be the primary source of light used to photoirradiate animals for photodynamic therapy studies. The intratumor temperature increases should be documented at multiple positions, to determine the amount of thermotolerance which can be induced. When photodynamic therapy is followed with a subsequent hyperthermia treatment, this induced thermotolerance can then be taken into consideration.     

The Effects of Thrombocytopenia on Vessel Stasis and Macromolecular Leakage after Photodynamic Therapy Using Photofrin

Abstract— Several studies have reported thrombus formation and/or the release of specific vasoactive eicosanoids, suggesting that platelet activation or damage after photodynamic therapy (PDT) may contribute to blood flow stasis. The role of circulating platelets on blood flow stasis and vascular leakage of macromolecules during and after PDT was assessed in an intravital animal model. Sprague-Daw-ley rats bearing chondrosarcoma on the right hind limb were injected intravenously (i.v.) with 25 mg/kg Photofrin 24 h before light treatment of 135 J/cm² at 630 nm. Thrombocytopenia was induced in animals by administration of 3.75 mg/kg of rabbit anti-rat platelet antibody i.v. 30 min before the initiation of the light treatment. This regimen reduced circulating platelet levels from 300000/mm³ to 20000/mm³. Reductions in the luminal diameter of the microvasculature in normal muscle and tumor were observed in control animals given Photofrin and light. Venule leakage of macromolecules was noted shortly after the start of light treatment and continued throughout the period of observation. Animals made thrombocytopenic showed none of these changes after PDT in either normal tissues or tumor. The lack of vessel response correlated with the absence of thromboxane release in blood during PDT. These data suggest that platelets and eicosanoid release are necessary for vessel constriction and blood flow stasis after PDT using Photofrin.     

OPTICAL PROPERTIES OF EXPERIMENTAL PROSTATE TUMORS in vivo

The optical properties of tumor tissue provide important information for optimizing treatment plans in photodynamic therapy, especially when intertitial application by multiple fibers is planned. Near infrared light, required to activate novel photosensitizers, should facilitate improved light penetrance of tumor tissue compared with 630 nm light used for activating Photofrin II. We have measured light energy fluence rates for 630 and 789 nm light along radial tracks from a single laterally diffusing optical fiber centrally implanted into Dunning R3327-AT and R3327-H rat tracks from a single laterally diffusing optical fiber centrally implanted into Dunning R3327-AT and R3327-H rat prostate tumors in anesthetized rats. A total of 20 R3327-AT and 10 R3327-H tumors were used in this study with volumes from 2.6 to 13.3 cm³. Light track data were analyzed by an empirical model that described light attenuation. At 630 nm, light attenuation coefficients (LAC) were T1.9 × higher than those at 789 nm for both tumors with the well-differentiated, well-perfused tumor (R3327-H) attenuating to a greater extent than did the rapidly growing anaplastic tumor (R3327-AT). The intertumor variation of LAC was greater than the spatial variations observed within individual tumors. LAC were a function of tumor volume for only 630 nm light in the R3327-AT tumors.     

A Comparative Analysis of Silicon Phthalocyanine Photosensitizers for in vivo Photodynamic Therapy of RIF-1 Tumors in C3H Mice

Photofrin® photodynamic therapy (PDT) has recently received FDA approval for the palliative treatment of to-tally and partially obstructing esophageal malignancies. However, there is a need for new PDT photosensitizers because Photofrin has a number of undesirable features. The purpose of this study was to evaluate the efficacy of four amine-bearing silicon phthalocyanines—Pc4, Pc10, Pc12 and Pc18—as potential PDT photosensitizers. Equimolar concentrations of these Pc were found to be highly effective at causing the regression of RIF-1 tumors trans-planted to C3H/HeN mice. The amount of Pc4 necessary to cause an equivalent amount of tumor regression in this model system was substantially less than the amount of Photofrin. The cutaneous phototoxicity of the silicon Pc photosensitizer was assessed by the utilization of the murine ear-swelling model. When C3H mice were exposed to 167 J/cm² of polychromatic visible light from a UVB-filtered solar simulator, which emitted UV radiation and visible light above 320 nm, the Pc produced little, if any, cutaneous photosensitivity. These results indicate that Pc4, Pc10, Pc12 and Pc18 are at least as effective as Photofrin in PDT protocols, while at the same time addressing many of the drawbacks of Photofrin.     

Distribution and pharmacokinetics of Photofrin in human bile duct cancer

Prognosis of patients with bile duct tumors is mostly poor due to late diagnosis and a lack of adequate curative and palliative treatment modalities. To evaluate the potential of photodynamic therapy (PDT) as a novel and alternative treatment approach, we have investigated the uptake and tumor-specific localization of the photosensitizer Photofrin in human biliary tract neoplasms. We have quantified the distribution and the pharmacokinetics of Photofrin in normal and tumor tissue biopsies of the human bile duct by quantitative fluorescence microscopy and digital image analysis of cryosections. Fluorescence intensities (expressed as a percentage of a standard) are 19.0 +/- 11.4% and 25.2 +/- 12.7% for tumors and 10.9 +/- 2.9% and 13.2 +/- 9.1% (mean +/- SD) for normal bile duct tissue at 24 h (n = 5) and 48 h (n = 8) after Photofrin administration (2 mg kg-1 i.v.), respectively, and decrease afterwards in normal bile duct tissue over the period of investigation (4-35 days). The ratios of fluorescence in tumor versus normal tissue are found to be 1.7 +/- 0.7 and 2.3 +/- 1.2 (mean +/- SD) at days one and two after Photofrin administration, respectively. Thus, Photofrin preferentially accumulates in bile duct neoplasms, reaching peak values during the first two days. These data suggest that laser irradiation should be performed within this period after Photofrin injection to achieve tumor selectivity of PDT for effective treatment of bile duct carcinoma.     

Photodynamic Therapy of Human Glioma (U87) in the Nude Rat

Abstract— We measured the response of normal brain and the human U87 glioma implanted in the brain of rats (n = 65) to photodynamic therapy (PDT) using Photofrin as the sensitizer. Normal brain and U87 tumor implanted within brain of athymic (nude) rats were subjected to PDT (12.5 mg/kg of Photofrin) at increasing optical energy doses (35 J/cm², 140 J/cm², 280 J/cm²) of 632 nm light. Photofrin concentration in tumor, brain adjacent to tumor and normal brain were measured in a separate population of rats. Twenty-four hours after PDT, the brains were removed, sectioned, stained with hematoxylin and eosin (H&E), and the volumes of the PDT-induced lesion measured. Photofrin concentration in tumor greatly exceeded that of normal brain and brain adjacent to tumor (>20×). Both normal brain and U87 tumor exhibited superficial tissue damage with PDT at 35 J/cm². However, both normal and tumor-implanted brain exhibited tissue damage with increasing optical dose. A heterogeneous pattern of pannecrosis along with a uniform volume of pannecrosis was detected in the tumor. In contrast, normal brain exhibited a uniform sharply demarcated volume of necrosis. Our data indicate that the U87 human brain tumor model and the normal brain in the athymic rat are sensitive to PDT and Photofrin with an optical dose-dependent response to treatment.     

Hyperoxygenation enhances the tumor cell killing of photofrin-mediated photodynamic therapy

Tumor hypoxia, either preexisting or as a result of oxygen depletion during photodynamic therapy (PDT) light irradiation, can significantly reduce the effectiveness of PDT-induced cell killing. To overcome tumor hypoxia and improve tumor cell killing, we propose using supplemental hyperoxygenation during Photofrin-PDT. The mechanism for the tumor cure enhancement of the hyperoxygenation-PDT combination is investigated using an in vivo-in vitro technique. A hypoxic tumor model was established by implanting mammary adenocarcinoma in the hind legs of mice. Light irradiation (200 J/cm2 at either 75 or 150 mW/cm2), under various oxygen supplemental conditions (room air, carbogen, 100% normobaric or hyperbaric oxygen), was delivered to animals that received 12.5 mg/kg Photofrin 24 h before light irradiation. Tumors were harvested at various time points after PDT and grown in vitro for colony formation analysis. Treated tumors were also analyzed histologically. The results show that when PDT is combined with hyperoxygenation, the hypoxic condition could be improved and the cell killing rate at various time points after PDT could be significantly enhanced over that without hyperoxygenation, suggesting an enhanced direct and indirect cell killing associated with high-concentration oxygen breathing. This study further confirms our earlier observation that when a PDT treatment is combined with hyperoxygenation it can be more effective in controlling hypoxic tumors.     

DISTRIBUTION AND ELIMINATION OF PHOTOFRIN II IN MICE

The distribution and elimination of [14C]PII, the radioisotopically-labeled equivalent of the mixture of porphyrins known as Photofrin II used in the photodynamic treatment of solid tumors, were determined in tumor-free and SMT-F tumor-bearing DBA/2 Ha-DD mice. Following i.p. injection, drug was absorbed from the peritoneum with a half-life of about 1 h; elimination from plasma was rapid, declining about 1.4 logs in concentration over 48 h following i.v. administration. However, some [14C]-activity was still detectable after 75 days. Normal tissues take up the drug within about 7.5 h after administration, with peak concentrations distributed as follows: liver, adrenal gland, urinary bladder greater than pancreas, kidney, spleen greater than stomach, bone, lung, heart greater than muscle much greater than brain. Only skeletal muscle, brain, and skin located contralaterally to subcutaneously implanted SMT-F tumors had peak [14C]-activities lower than tumor tissue; skin overlying SMT-F tumors showed concentrations not significantly different (P greater than 0.3) from tumor. After 75 days all tissues examined retained some fraction of [14C]-activity, ranging from 16% for kidney to 61% for spleen, of the initial peak tissue levels. The primary route of elimination of Photofrin II was through the bile-gut pathway, with greater than 59% of the administered [14C]-activity recovered in the feces, and only about 6% in the urine, over 192 h. HPLC analyses of fecal extracts showed that mostly monomeric and other low molecular weight porphyrin components of Photofrin II were eliminated. The higher molecular weight oligomeric fractions of Photofrin II were retained in liver and spleen up to 14 days after injection.     

Subcellular Localization of Photofrin and Aminolevulinic Acid and Photodynamic Cross-Resistance in Vitro in Radiation-Induced Fibrosarcoma Cells Sensitive or Resistant to Photofrin-Mediated Photodynamic Therapy

Abstract— The subcellular and, specifically, mitochondrial localization of the photodynamic sensitizers Photofrin and aminolevulinic acid (ALA)-induced protoporphyrin-IX (PpIX) has been investigated in vitro in radiation-induced fibrosarcoma (RIF) tumor cells. Comparisons were made of parental RIF-1 cells and cells (RIF-8A) in which resistance to Photofrin-mediated photodynamic therapy (PDT) had been induced. The effect on the uptake kinetics of Photofrin of coincubation with one of the mitochondria-specific probes 10N-Nonyl acridine orange (NAO) or rhodamine-123 (Rh-123) and vice versa was examined. The subcellular colocalization of Photofrin and PpIX with Rh-123 was determined by double-label confocal fluorescence microscopy. Clonogenic cell survival after ALA-mediated PDT was determined in RIF-1 and RIF-8A cells to investigate cross-resistance with Photofrin-mediated PDT. At long (18 h) Photofrin incubation times, stronger colocalization of Photofrin and Rh-123 was seen in RIF-1 than in RIF-8A cells. Differences between RIF-1 and RIF-8A in the competitive mitochondrial binding of NAO or Rh-123 with Photofrin suggest that the inner mitochondrial membrane is a significant Photofrin binding site. The differences in this binding may account for the PDT resistance in RIF-8A cells. With ALA, the peak accumulations of PpIX occurred at 5 h for both cells, and followed a diffuse cytoplasmic distribution compared to mitochondrial localization at 1 h ALA incubation. There was rapid efflux of PpIX from both RIF-1 and RIF-8A. As with Photofrin, ALA-induced PpIX exhibited weaker mitochondrial localization in RIF-8A than in RIF-1 cells. Clonogenic survival demonstrated cross-resistance to incubation in PpIX but not to ALA-induced PpIX, implying differences in mitochondrial localization and/or binding, depending on the source of the PpIX within the cells.     

RESISTANCE TO PHOTODYNAMIC THERAPY IN RADIATION INDUCED FIBROSARCOMA-1 and CHINESE HAMSTER OVARY-MULTI-DRUG RESISTANT CELLS in vitro

A degree of resistance to photodynamic therapy (PDT) has been induced in radiation-induced fibrosarcoma-1 (RIF-1) tumor cells by repeated photodynamic treatment with Photofrin (4 or 18 h incubation) in vitro to the 0.1-1% survival level, followed by regrowth from single surviving colonies. The resistance is shown as increased cell survival in the strain designated RIF-8A, compared to the wild-type RIF-1 cells, when exposed to increasing Photofrin concentration for 18 h incubation and fixed light exposure. No difference was found between RIF-1 and RIF-8A in the uptake of Photofrin per unit cell volume at 18 h incubation. Resistance to PDT was also observed in Chinese hamster ovary-multi-drug resistant (CHO-MDR) cells compared to the wild-type CHO cells, possibly associated with decreased cellular concentration of Photofrin in the former. By contrast, the PDT-resistant RIF-8A cells did not show any cross-resistance to Adriamycin, nor was there any significant drug concentration difference between RIF-1 and RIF-8A. These findings suggest that different mechanisms are responsible for PDT-induced resistance and multi-drug resistance.     

DRUG AND LIGHT DOSE DEPENDENCE OF PHOTODYNAMIC THERAPY: A STUDY OF TUMOR CELL CLONOGENICITY AND HISTOLOGIC CHANGES

Abstract— The dependence of photodynamic therapy (PDT) on changes in drug and light doses was determined in C3H/HeJ mice bearing the RIF tumor. Measurements of tumor clonogenicity were determined 24 h after PDT over a range of drug and light doses. Representative histological samples were prepared at each of these doses. Both the drug and light dose dependence experiments showed an exponential decrease in clonogenicity after an initial shoulder region. Reciprocity of drug and light dose was established from those clonogenicity curves. Histological examination of tumors gave information concerning the localization of gross damage within tumors. Increases of light dose in PDT were shown to extend the depth of necrosis within tumors. Increases of drug dose produced enlargements in the area of necrotic spots produced by PDT     

Protection of murine foot tissue and transplantable tumor against Photofrin-II-mediated photodynamic sensitization with WR-2721

Four thiol-containing compounds, WR-2721, WR-149024, WR-168643 and WR-361, were compared as photoprotectors of murine feet. The protector doses were the maximal tolerated intraperitoneal doses, administered 24 h after injection of Photofrin II and 15 min before illumination with 630-nm laser light. While all four compounds were effective, only WR-2721 demonstrated a statistically significant attenuation of phototoxicity. WR-2721 was found to protect SMT-F tumors in the same mouse strain, using tumor growth delay and short-term control as endpoints. A comparison of the dose modification factors for foot and tumor responses indicated no therapeutic advantage in using WR-2721 during photodynamic treatment of these two tissues.     

Interaction between photodynamic therapy and BCG immunotherapy responsible for the reduced recurrence of treated mouse tumors

Subcutaneous mouse EMT6 tumors were treated by individual or combined regimens of a single Bacillus Calmette-Guérin (BCG) vaccine administration and photodynamic therapy (PDT). Six clinically relevant photosensitizers characterized by different action mechanisms were used: Photofrin, benzoporphyrin derivative, tetra(m-hydroxyphenyl)chlorin (foscan), mono-L-aspartylchlorin e6, lutetium texaphyrin or zinc phthalocyanine. Irrespective of the type of photosensitizer used, the optimized BCG protocols improved the cure rate of PDT-treated tumors. This indicates that the interaction does not take place during the early phase of tumor ablation but at later events involved in preventing tumor recurrence. Beneficial effects on tumor cure were observed even when the BCG injection was delayed to 7 days after PDT. The accumulation of activated myeloid cells that markedly increases in tumors treated by Photofrin-based PDT was not additionally affected by BCG treatment. However, the incidence of immune memory T cells in tumor-draining lymph nodes that almost doubled at 6 days after Photofrin-PDT further increased close to three-fold with adjuvant BCG. This suggests that BCG immunotherapy amplifies the T-lymphocyte-mediated immune response against PDT-treated tumors. Since both these modalities are established for the treatment of superficial bladder carcinomas, use of their combination for this condition should be clinically tested.