The value of serum alpha-N-acetylgalactosaminidase measurement for the assessment of tumour response to radio- and photodynamic therapy.

Serum activity of alpha-N-acetylgalactosaminidase (NaGalase), the extracellular matrix-degrading enzyme that appears to be produced exclusively by cancer cells, was measured in mice bearing SCCVII tumours (squamous cell carcinoma). The NaGalase levels in these mice increased with time of tumour growth and were directly proportional to tumour burden. After exposure of SCCVII tumours to a single X-ray dose of 20 Gy, the serum NaGalase levels gradually decreased during the first 10 days after treatment (to approximately one-third of the initial value) and then began to increase. The decrease in serum NaGalase activity was more rapid after the treatment of SCCVII and EMT6 tumours by photodynamic therapy (PDT) and was dependent on the PDT dose. The treatments (based on photosensitizers Photofrin or mTHPC) that were fully curative resulted in the reduction of NaGalase activity to background levels within 2 or 3 days after PDT. A slower decrease in NaGalase activity was found after PDT treatments that attain an initial tumour ablation but are not fully curative. The regrowth of PDT-treated SCCVII tumours was preceded by an increase in serum NaGalase levels, which was detected as early as 8 days before the visible tumour reappearance. These findings ascertain the validity of serum NaGalase measurement for the assessment of tumour response to different treatments and support the concept that the NaGalase measurement could serve as a diagnostic and prognostic index that might allow oncologists to design the dosage or nature of treatment.     

Induction of haem oxygenase-1 nitric oxide and ischaemia in experimental solid tumours and implications for tumour growth.

Induction of haem oxygenase-1 (HO-1) as well as nitric oxide (NO) biosynthesis during tumour growth was investigated in an experimental solid tumour model (AH136B hepatoma) in rats. An immunohistochemical study showed that the inducible isoform of NO synthase (iNOS) was localized in monocyte-derived macrophages, which infiltrated interstitial spaces of solid tumour, but not in the tumour cells. Excessive production of NO in the tumour tissue was unequivocally verified by electron spin resonance spectroscopy. Tumour growth was moderately suppressed by treatment with either Nomega-nitro-L-arginine methyl ester (L-NAME) or S-methylisothiourea sulphate (SMT). In contrast, HO-1 was found only in tumour cells, not in macrophages, by in situ hybridization for HO-1 mRNA. HO-1 expression in AH136B cells in culture was strongly enhanced by an NO (NO+) donor S-nitroso-N-acetyl penicillamine. HO-1 mRNA expression in the solid tumour in vivo decreased significantly after treatment with low doses of NOS inhibitors such as L-NAME and SMT (6-20 mg kg(-1)). However, the level of HO-1 mRNA in the solid tumour treated with higher doses of NOS inhibitor was similar to that of the solid tumour without NOS inhibitor treatment. Strong induction of HO-1 was also observed in solid tumours after occlusion or embolization of the tumour-feeding artery, indicating that ischaemic stress which may involve oxidative stress triggers HO-1 induction in the solid tumour. Lastly, it is of great importance that an HO inhibitor, zinc protoporphyrin IX injected intra-arterially to the solid tumour suppressed the tumour growth to a great extent. In conclusion, HO-1 expression in the solid tumour may confer resistance of tumour cells to hypoxic stress as well as to NO-mediated cytotoxicity.     

Ischaemia-reperfusion injury in photodynamic therapy-treated mouse tumours

Prompted by the observation of ischaemia development during the treatment of tumours by photodynamic therapy (PDT) that is typically followed by a restoration of tumour blood flow and by the indications of secondary superoxide generation after PDT, we aimed in this study to obtain evidence of the induction of ischaemia-reperfusion (I/R) injury in PDT-treated tumours. Using subcutaneous mouse FsaR fibrosarcoma model and Photofrin-based PDT treatment, we have examined the activity of xanthine oxidase (XO, a key enzyme in the I/R injury development) in tumours before and after the therapy. Compared to the levels in nontreated tumours, there was a five-fold increase in the activity of this enzyme in tumours excised immediately after PDT. This burst of elevated XO activity declined rapidly, returning to the pretreatment levels within the next 30 min. Visible reflectance spectroscopy confirmed the occurrence of a PDT-induced strong but temporary reduction in tumour oxygenation. The administration of XO inhibitor oxypurinol prevented this PDT-induced rise in XO activity. The oxypurinol treatment also decreased the extent of neutrophil accumulation in PDT-treated tumours and reduced the level of PDT-mediated cures. These results demonstrate the induction of I/R injury in PDT-treated tumours, and show that it can contribute to the therapy outcome. Since I/R injury is a well-recognised proinflammatory insult, we suggest that its induction in PDT-treated tumours promotes the development of inflammatory response that has become established as a key element of the antitumour effect of PDT.     

Changes in perfusion of mouse tumours after photodynamic therapy

The influence of photodynamic therapy (PDT) on vascular perfusion was investigated in 2 s.c. mouse tumours, a radiation-induced fibrosarcoma (RIFI) and a squamous-cell carcinoma (SCCVII). The ⁸⁶Rb extraction technique was used to measure changes in perfusion relative to cardiac output at various intervals after interstitial PDT. Control groups showed that vascular perfusion in the RIFI tumours decreased with increasing tumour size. For both tumours, of constant size, vascular perfusion decreased to less than 10% of control values within 5 min after high PDT doses. Significant decreases in vascular perfusion were also seen after lower, sub-curative doses. Thereafter there was slow recovery towards control levels. Photofrin given at shorter intervals before illumination generally resulted in even larger decreases in tumour perfusion, and slower recovery. Comparison of tumour perfusion measurements after PDT with tumour response revealed an inverse correlation with tumour growth delay both for the RIFI and for the SCCVII tumours. PDT with sub-curative light doses appears to decrease vascular perfusion in the RIFI and SCCVII for a period of at least 24 hr. The most severe reductions in tumour blood flow were associated with the longest regrowth delays, indicating a major role of vascular damage in tumour response to PDT.     

Macrophage-directed immunotherapy as adjuvant to photodynamic therapy of cancer.

The effect of Photofrin-based photodynamic therapy (PDT) and adjuvant treatment with serum vitamin D3-binding protein-derived macrophage-activating factor (DBPMAF) was examined using a mouse SCCVII tumour model (squamous cell carcinoma). The results show that DBPMAF can markedly enhance the curative effect of PDT. The most effective DBPMAF therapy consisted of a combination of intraperitoneal and peritumoral injections (50 and 0.5 ng kg-1 respectively) administered on days 0, 4, 8 and 12 after PDT. Used with a PDT treatment curative to 25% of the treated tumours, this DBPMAF regimen boosted the cures to 100%. The DBPMAF therapy alone showed no notable effect on the growth of SCCVII tumour. The PDT-induced immunosuppression, assessed by the evaluation of delayed-type contact hypersensitivity response in treated mice, was greatly reduced with the combined DBPMAF treatment. These observations suggest that the activation of macrophages in PDT-treated mice by adjuvant immunotherapy has a synergistic effect on tumour cures. As PDT not only reduces tumour burden but also induces inflammation, it is proposed that recruitment of the activated macrophages to the inflamed tumour lesions is the major factor for the complete eradication of tumours.     

Induction of immune cell infiltration into murine SCCVII tumour by photofrin-based photodynamic therapy.

Cellular populations in the squamous cell carcinoma SCCVII, growing in C3H/HeN mice given Photofrin, were examined at various time intervals during the photodynamic light treatment and up to 8 h later. Cell populations present within excised tumours were identified by monoclonal antibodies directed against cell type-specific membrane markers using a combination of the indirect immunoperoxidase and Wright staining or by flow cytometry. Photofrin-based photodynamic therapy (PDT) induced dramatic changes in the level of different cellular populations contained in the treated tumour. The most pronounced was a rapid increase in the content of neutrophils, which increased 200-fold within 5 min after the initiation of light treatment. This was followed immediately by an increase in the levels of mast cells, while another type of myeloid cells, most likely monocytes, invaded the tumour between 0 and 2 h after PDT. The examination of cytolysis of in vitro cultured SCCVII tumour cells mediated by macrophages harvested from the SCCVII tumour revealed a pronounced increase in the tumoricidal activity of tumour-associated macrophages isolated at 2 h post PDT. It seems, therefore, that the PDT-induced acute inflammatory infiltration of myeloid cells into the treated tumour is associated with functional activation of immune cells.     

Photofrin accumulation in malignant and host cell populations of various tumours.

Photofrin accumulation in malignant and host cell populations of various tumours was studied by flow cytometry analysis of cells dissociated from the tumour tissue. The transplantable mouse tumour models included in this analysis were sarcomas EMT6, RIF, KHT and FsaN, Lewis lung carcinoma, SCCVII squamous cell carcinoma (SCC) and slowly growing moderately differentiated AT17 SCC. An example of spontaneous mouse adenocarcinoma was also examined. Staining with specific monoclonal antibodies was used to identify the various cell populations present in these tumours. The main characteristic of Photofrin cellular accumulation was a very high photosensitiser content found exclusively in a subpopulation of tumour-associated macrophages (TAMs). Photosensitiser levels similar to or lower than in malignant cells were observed in the remaining TAMs and other tumour-infiltrating host cells. Photofrin accumulation in malignant cells was not equal in all tumour models, but may have been affected by tumour blood perfusion/vascularisation. Results consistent with the above findings were obtained with SCC of buccal mucosa induced by 9,10-dimethyl-1,2-benzanthracene in Syrian hamsters. The TAM subpopulation that accumulates by far the highest cellular Photofrin levels in tumours is suggested to be responsible for the tumour-localised photosensitiser fluorescence.     

Photodynamic therapy-generated vaccines: relevance of tumour cell death expression

Recent investigations have established that tumour cells treated in vitro by photodynamic therapy (PDT) can be used for generating potent vaccines against cancers of the same origin. In the present study, cancer vaccines were prepared by treating mouse SCCVII squamous cell carcinoma cells with photosensitiser chlorin e6-based PDT and used against poorly immunogenic SCCVII tumours growing in syngeneic immunocompetent mice. The vaccine potency increased when cells were post-incubated in culture after PDT treatment for 16 h before they were injected into tumour-bearing mice. Interfering with surface expression of phosphatidylserine (annexin V treatment) and apoptosis (caspase inhibitor treatment) demonstrated that this post-incubation effect is affiliated with the expression of changes associated with vaccine cell death. The cured mice acquired resistance to re-challenge with the same tumour, while the engagement of cytotoxic T lymphocytes was demonstrated by detection of high numbers of degranulating CD8+ cells in vaccinated tumours. The vaccines prepared from ex vivo PDT-treated SCCVII tumour tissue were also highly effective, implying that surgically removed tumour tissue can be directly used for PDT vaccines. This opens attractive prospects for employing PDT vaccines tailored for individual patients targeting specific antigens of the patient's tumour.     

Potentiation of photodynamic therapy of cancer by complement: the effect of gamma-inulin

Host response elicited by photodynamic therapy (PDT) of cancerous lesions is a critical contributor to the clinical outcome, and complement system has emerged as its important element. Amplification of complement action was shown to improve tumour PDT response. In search of a clinically relevant complement activator for use as a PDT adjuvant, this study focused on gamma-inulin and examined its effects on PDT response of mouse tumours. Intralesional gamma-inulin (0.1 mg mouse(-1)) delivered immediately after PDT rivaled zymosan (potent classical complement activator) in delaying the recurrence of B16BL6 melanomas. This effect of gamma-inulin was further enhanced by IFN-gamma pretreatment. Tumour C3 protein levels, already elevated after individual PDT or gamma-inulin treatments, increased much higher after their combination. With fibrosarcomas MCA205 and FsaR, adjuvant gamma-inulin proved highly effective in reducing recurrence rates following PDT using four different photosensitisers (BPD, ce6, Photofrin, and mTHPC). At 3 days after PDT plus gamma-inulin treatment, over 50% of cells found at the tumour site were CTLs engaged in killing specific targets via perforin-granzyme pathway. This study demonstrates that gamma-inulin is highly effective PDT adjuvant and suggests that by amplifying the activation of complement system, this agent potentiates the development of CTL-mediated immunity against PDT-treated tumours.     

Recovery from potentially lethal damage after X-irradiation in three experimental tumors in mice

Potentially lethal damage repair or recovery (PLDR) after X-irradiation was examined in three experimental murine tumors, EMT6 sarcoma, SCCVII carcinoma and RIF1 sarcoma, by an in vivo-in vitro assay. All tumors were transplanted subcutaneously (SC) and, in the case of EMT6, also intradermally (id), in the thighs of syngeneic mice. Apparent PLDR was observed at high dose levels in the id and sc EMT6 sarcomas and in the sc SCCVII carcinoma, but not in the sc RIF1 sarcoma. In both id EMT6 and SCCVII tumors, PLDR appeared to be complete within 9 hr after X-irradiation. Dose-modifying factors due to PLDR evaluated at the surviving fraction of 0.01 were 1.34, 1.15, 1.24 and 0.97 for id EMT6, sc EMT6, SCCVII and RIF1 tumors, respectively. The amount of PLDR correlated inversely with the velocity of tumor growth in vivo, suggesting that slowly growing tumors might be proficient in PLDR. Although PLDR in id EMT6 and SCCVII tumors was evident in the dose range above 15 Gy, the capacity to recovery from PLD became smaller at lower doses; in SCCVII tumors, this phenomenon was negligible below 7.5 Gy. PLDR did not seem to be a major factor influencing the radiocurability of these murine tumors, especially at low dose levels.     

Cellular levels of photosensitisers in tumours: the role of proximity to the blood supply.

Flow cytometry using the tumour perfusion probe Hoechst 33342 was employed to examine the distribution of photosensitisers in tumour cells located at different distances from the blood supply. Two tumour models, the SCCVII squamous cell carcinoma and FsaR fibrosarcoma growing in C3H/HeN mice, were used in the experiments. Among the photosensitisers tested, only BPD (benzoporphyrin derivative monoacid) exhibited uniform distribution in tumour cells irrespective of their distance from the vasculature. In this respect, 5-aminolaevulinic acid (i.e. its metabolite protoporphyrin IX), di- and tetrasulphonated aluminium phthalocyanines (A1PcS2 and AlPcS4), di- and tetrasulphonated tetraphenylporphines (TPPS2 and TPPS4), Photofrin and bacteriochlorophyll-a (i.e. its metabolite bacteriopheophytin-a) followed BPD in decreasing order in their efficacy of accumulation in tumour cells remote from the blood supply. This photosensitiser property appeared not to depend on tumour type, tumour size, route of photosensitiser administration, time after the administration, photosensitiser lipophilicity or on the presence of host cell infiltrate in the tumour. Following treatment with photodynamic therapy (PDT) in vivo, tumour cells were sorted based on their blood vessel proximity and their survival was determined by colony formation assay. The data demonstrate that the direct killing of tumour cells by Photofrin- and A1PcS2-based PDT decreases with increasing distance of the cells from the blood supply.     

Comparison between the comet assay and pimonidazole binding for measuring tumour hypoxia

Pimonidazole is finding increasing use in histochemical analyses of hypoxia in tumours. Whether it can identify every hypoxic cell in a tumour, and whether the usual subjective criteria used to define 'positive' cells are optimal, are less certain. Therefore, our aim was to develop an objective flow cytometry procedure for quantifying pimonidazole binding in tumours, and to validate this method by using a more direct indicator of radiobiologic hypoxia, the comet assay. SCCVII tumours in C3H mice were analysed for pimonidazole binding using flow cytometry and an iterative curve-fitting procedure, and the results were compared to the comet assay for the same cell suspensions. On average, cells defined as anoxic by flow analysis (n = 43 tumours) bound 10.8 +/- 0.95 times more antibody than aerobic cells. In samples containing known mixtures of aerobic and anoxic cells, hypoxic fractions as low as 0.5% could easily be detected. To assess the flow cytometry assay under a wider range of tumour oxygen contents, mice were injected with hydralazine to reduce tumour blood flow, or allowed to breathe various gas mixtures during the 90 min exposure to pimonidazole. Hypoxic fraction estimated by the pimonidazole binding method agreed well with the hypoxic fraction measured using the comet assay in SCCVII tumours (r2 = 0.87, slope = 0.98), with similar results in human U87 glioma cells and SiHa cervical carcinoma xenografts. We therefore conclude that this objective analysis of pimonidazole labelling by flow cytometry gives a convenient and accurate estimate of radiobiological hypoxia. Preliminary analyses of biopsies from 3 patients given 0.5 g m-2 pimonidazole also suggest the suitability of this approach for human tumours.     

The Influence of Nitric Oxide on Tumour Vascular Tone

Acetylcholine and sodium nitroprusside, which vasodilate via release of NO by endothelium-dependent and endothelium-independent mechanisms respectively, had little effect on tumour vascular resistance when administered to tissue-isolated tumours perfused in their normal state. However, under phenylephrine-induced vasoconstriction, sodium nitroprusside induced vasodilation whilst acetylcholine induced a small vasoconstriction. Phenylephrine itself induced an oscillatory change in tumour perfusion pressure. The nitric oxide synthase (NOS) inhibitor N^Ω-nitro-L-arginine (L-NNA) caused a dose-dependent increase in vascular resistance in ex vivo perfused tumours which was greater than that in normal perfused hindlimbs. Systemic administration of L-NNA caused a 50% decrease in tumour blood flow which was a larger effect than in any of the normal tissues studied except spleen and skeletal muscle. Modification of NOS activity in tumours is a promising means for selective tumour blood flow modification. Investigation of endothelium-dependent versus endothelium-independent methods for modifying tumour blood flow may provide methods for further selectivity.     

A novel combretastatin A-4 derivative,AC7700, strongly stanches tumour blood flow and inhibits growth of tumours developing in various tissues and organs

In a previous study, we used subcutaneous LY80 tumours (a subline of Yoshida sarcoma), Sato lung carcinoma, and methylcholanthrene-induced primary tumours, to demonstrate that a novel water-soluble combretastatin A-4 derivative, AC7700, abruptly and irreversibly stopped tumour blood flow. As a result of this interrupted supply of nutrients, extensive necrosis was induced within the tumour. In the present study, we investigated whether AC7700 acts in the same way against solid tumours growing in the liver, stomach, kidney, muscle, and lymph nodes. Tumour blood flow and the change in tumour blood flow induced by AC7700 were measured by the hydrogen clearance method. In a model of cancer chemotherapy against metastases, LY80 cells (2x10(6)) were injected into the lateral tail vein, and AC7700 at 10 mg x kg(-1) was injected i.v. five times at intervals of 2 days, starting on day 7 after tumour cell injection. The number and size of tumours were compared with those in the control group. The change in tumour blood flow and the therapeutic effect of AC7700 on microtumours were observed directly by using Sato lung carcinoma implanted in a rat transparent chamber. AC7700 caused a marked decrease in the tumour blood flow of all LY80 tumours developing in various tissues and organs and growth of all tumours including lymph node metastases and microtumours was inhibited. In every tumour, tumour blood flow began to decrease immediately after AC7700 administration and reached a minimum at approximately 30 min after injection. In many tumour capillaries, blood flow completely stopped within 3 min after AC7700 administration. These results demonstrate that AC7700 is effective for tumours growing in various tissues and organs and for metastases. We conclude that tumour blood flow stanching induced by AC7700 may become an effective therapeutic strategy for all cancers, including refractory cancers because the therapeutic effect is independent of tumour site and specific type of cancer.     

Nitric oxide-mediated promotion of mammary tumour cell migration requires sequential activation of nitric oxide synthase,guanylate cyclase and mitogen-activated protein kinase

Using clonal derivatives of spontaneous mammary tumours in C3H/HeJ mice, we had earlier shown that tumour-derived nitric oxide (NO), resulting from endothelial type (e) NO synthase (NOS) expression by tumour cells, promoted tumour growth and metastasis by multiple mechanisms: stimulation of tumour cell invasiveness, migration and angiogenesis. Our present study examined the signaling mechanisms underlying NO-mediated promotion of tumour cell migration in a highly metastatic and high eNOS-expressing C3H/HeJ mammary tumour cell line, C3L5. C3L5 cell migration was reduced in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor) in a concentration-dependent manner and restored in the additional presence of excess L-arginine (NOS substrate), confirming a migration-promoting role of endogenous NO. Migratory capacity of C3L5 cells was reduced after treatment with the guanylate cyclase (GC) inhibitor 1-H-[1,2,4]oxadiaxolo[4,3-a]quinolalin-1-one (ODQ) and restored in the additional presence of 8-bromoguanosine 3'5'-cyclic monophosphate (8-Br cGMP, cGMP analogue), demonstrating a pivotal role for GC in C3L5 cell migration. Mitogen-activated protein kinase kinase (MAPKK; MEK) inhibitor, UO126, blocked migration, demonstrating MEK involvement in C3L5 cell migration. Furthermore, both ODQ and UO126 blocked migration-restoring effects of L-arginine in L-NAME-treated cells, indicating that GC and MAPK pathways are required for endogenous NO-mediated migratory responses. Similarly, L-NAME reduced and additional treatment with excess L-arginine or sodium nitroprusside (SNP, NO donor) stimulated phosphorylation of extracellular signal-regulated kinases (ERK(1/2)), demonstrating a role for endogenous and exogenous NO in ERK(1/2) activation. ODQ inhibited ERK(1/2) activation, whereas 8-Br cGMP stimulated ERK(1/2) phosphorylation in L-NAME-treated cells, indicating that cGMP is a downstream effector of NOS for ERK(1/2) activation. Finally, both ODQ and UO126 blocked the capacity of L-arginine to restore ERK(1/2) phosphorylation in L-NAME-treated cells, demonstrating that GC and MEK are both required for endogenous NO-mediated MAPK activation. Together, these results indicate sequential activation of NOS, GC and MAPK pathways in mediating signals for C3L5 cell migration, an essential step in invasion and metastasis. Since NOS activity is positively associated with human breast cancer progression, the present results are relevant for development of therapeutic modalities for this disease.     

Nitric oxide synthase inhibition results in synergistic anti-tumour activity with melphalan and tumour necrosis factor alpha-based isolated limb perfusions

Nitric oxide (NO) is an important molecule in regulating tumour blood flow and stimulating tumour angiogenesis. Inhibition of NO synthase by L-NAME might induce an anti-tumour effect by limiting nutrients and oxygen to reach tumour tissue or affecting vascular growth. The anti-tumour effect of L-NAME after systemic administration was studied in a renal subcapsular CC531 adenocarcinoma model in rats. Moreover, regional administration of L-NAME, in combination with TNF and melphalan, was studied in an isolated limb perfusion (ILP) model using BN175 soft-tissue sarcomas. Systemic treatment with L-NAME inhibited growth of adenocarcinoma significantly but was accompanied by impaired renal function. In ILP, reduced tumour growth was observed when L-NAME was used alone. In combination with TNF or melphalan, L-NAME increased response rates significantly compared to perfusions without L-NAME (0-64% and 0-63% respectively). An additional anti-tumour effect was demonstrated when L-NAME was added to the synergistic combination of melphalan and TNF (responses increased from 70 to 100%). Inhibition of NO synthase reduces tumour growth both after systemic and regional (ILP) treatment. A synergistic anti-tumour effect of L-NAME is observed in combination with melphalan and/or TNF using ILP. These results indicate a possible role of L-NAME for the treatment of solid tumours in a systemic or regional setting.     

CD8+ T cell-mediated control of distant tumours following local photodynamic therapy is independent of CD4+ T cells and dependent on natural killer cells

Cancer survival rates decrease in the presence of disseminated disease. However, there are few therapies that are effective at eliminating the primary tumour while providing control of distant stage disease. Photodynamic therapy (PDT) is an FDA-approved modality that rapidly eliminates local tumours, resulting in cure of early disease and palliation of advanced disease. Numerous pre-clinical studies have shown that local PDT treatment of tumours enhances anti-tumour immunity. We hypothesised that enhancement of a systemic anti-tumour immune response might control the growth of tumours present outside the treatment field. To test this hypothesis we delivered PDT to subcutaneous (s.c.) tumours of mice bearing both s.c. and lung tumours and monitored the growth of the untreated lung tumours. Our results demonstrate that PDT of murine tumours provided durable inhibition of the growth of untreated lung tumours. The inhibition of the growth of tumours outside the treatment field was tumour-specific and dependent on the presence of CD8(+) T cells. This inhibition was accompanied by an increase in splenic anti-tumour cytolytic activity and by an increase in CD8(+) T cell infiltration into untreated tumours. Local PDT treatment led to enhanced anti-tumour immune memory that was evident 40 days after tumour treatment and was independent of CD4(+) T cells. CD8(+) T cell control of the growth of lung tumours present outside the treatment field following PDT was dependent upon the presence of natural killer (NK) cells. These results suggest that local PDT treatment of tumours lead to induction of an anti-tumour immune response capable of controlling the growth of tumours outside the treatment field and indicate that this modality has potential in the treatment of distant stage disease.     

Photodynamic therapy using intravenous delta-aminolaevulinic acid-induced protoporphyrin IX sensitisation in experimental hepatic tumours in rats.

The efficacy of photodynamic therapy (PDT) using delta-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) sensitisation and laser light at 635 nm was investigated in the treatment of experimental hepatic tumours. The model of liver tumours was induced either by local inoculation or by administration of tumour cells through the portal vein in rats. ALA at a dose of 60 mg kg(-1) b.w. was intravenously administered 60 min before PDT. PpIX accumulation in tumour, normal liver and abdominal wall muscle was detected by means of laser-induced fluorescence (LIF). Laser Doppler imaging (LDI) was used to determine changes in the superficial blood flow in connection with PDT. Histopathological examinations were performed to evaluate the PDT effects on the tumour and the surrounding liver tissue, including pathological features in the microvascular system. The accumulation of PpIX, as monitored by LIF, showed high fluorescence intensities at about 635 nm in both the hepatic tumour tissue and normal liver and low values in the abdominal wall. LDI demonstrated that the blood flow in the treated tumour and its surrounding normal liver tissue decreased immediately after the PDT, indicating an effect on the vascular system. A large number of thrombi in the irradiated tumour were found microscopically 3 h after the PDT. The tumour growth rate showed a marked decrease when evaluated 3 and 6 days after the treatment. These results show that the ALA-PDT is effective in the inhibition of growth of experimental hepatic tumours.     

Variation in the hypoxic fraction among mouse tumors of different types, sizes, and sites

Radiobiologically hypoxic fractions of 4 experimental tumors (RIF1, B16, EMT6/KU, SCCVII) of various sizes implanted subcutaneously (sc) or intradermally (id) were compared under identical conditions by using the paired survival curve assay method. The two survival curves for tumors in air-breathing and asphyxiated mice were almost parallel for EMT6/KU and SCCVII tumors, but not for RIF1 and B16 tumors. Therefore, hypoxic fractions were estimated by fitting the best parallel lines to the two sets of survival data. The values for 10 mm-diameter sc tumors in the hind legs of syngeneic mice were 4.7% for RIF1, 4.5% for B16, 14% for EMT6/KU, and 8.5% for SCCVII. The variation of the values is quite small in spite of the variety of biological characteristics of these tumors. Histological examination revealed that EMT6/KU and B16 tumors contained large necrotic areas, while SCCVII and RIF1 had small areas of necrosis. Thus, the hypoxic fraction and the proportion of necrosis in the histological specimens were not clearly correlated. The values for 6 mm and 16 mm sc EMT6/KU tumors were 5.9 and 22%, respectively, while that for 6 mm id tumors was 16%. The values for 5 mm and 18 mm sc SCCVII tumors were 0.86 and 8.4%, respectively. These results indicated that: (1) id EMT6/KU tumors had a higher hypoxic fraction than sc ones of the same size; (2) the hypoxic fraction increased with increase in tumor size in EMT6/KU tumors, while it reached a plateau at a certain size in SCVII tumors.     

Enhancement of photodynamic therapy by mitomycin C: a preclinical and clinical study.

Photodynamic therapy (PDT) using Photofrin was used in combination with a hypoxic toxin (mitomycin C, MMC) to treat four patients with recurrent skin metastasis of a mammary carcinoma. In preclinical experiments an additive effect was found for the combination of MMC and PDT for treating subcutaneous RIF1 tumours in mice. When interstitial PDT was combined with a low dose of MMC (administered 15 min before illumination), the Photofrin dose or light dose could be reduced by a factor of 2 in order to obtain equivalent cure rate or growth delay. In the clinical pilot study, a low dose of Photofrin (0.75 mg kg-1) was used for PDT alone (superficial illumination) or combined with low-dose MMC (5 mg m-2). Different tumour areas were illuminated with or without a preceding infusion of MMC. Both tumour response and skin photosensitivity were scored. After 8-12 weeks of treatment, tumour cure could be achieved by administering light doses > or = 150 J cm-2 for PDT alone and similar effects were obtained when light doses of 75-87.5 J cm-2 were given after infusion with MMC. In all cases necrotic tissue of both tumour and surrounding skin was observed, which lasted for a mean of 5 months (range 2-20 months). Skin phototoxicity, tested by using a standardised illumination of skin patches on the back, lasted maximally 3 weeks. Three main conclusions could be drawn from these studies: (1) The enhanced effects of the combination of PDT and MMC observed in mouse tumours can be extrapolated to patients with mammary skin metastasis. (2) The combination of PDT and hypoxic toxins facilitates treatment by permitting lower doses of photosensitiser to be used (thereby reducing skin phototoxicity) or lower light doses (thereby reducing illumination times and allowing the possibility to treat larger tumour areas). (3) Restoration of skin after PDT in previously treated tumour areas (chemotherapy, radiation therapy and surgery) is very low.