David L. Liu

Pharmacokinetic studies on 5-aminolevulinic acid-induced protoporphyrin IX accumulation in tumours and normal tissues

Laser-induced fluorescence (LIF) for in vivo point monitoring and fluorescence microscopy incorporating a CCD camera were used to study the fluorescence distribution of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) in tumours. Fluorescence in a chemically-induced adenocarcinoma in the liver of rats and in an aggressive basal cell carcinoma in a patient were studied after intravenous injection of ALA at a dose of 30 mg/kg body weight. The LIF technique demonstrated slightly more ALA-induced PpIX fluorescence in the tumour than in the surrounding normal liver and abdominal muscle of rats. The visible parts of the human basal cell carcinoma exhibited strong ALA-induced fluorescence, while this fluorescence was much weaker in the necrotic areas of the tumour and in the surrounding normal skin.

Tumour vessel damage resulting from laser-induced hyperthermia alone and in combination with photodynamic therapy

This study examined tumour vessel injury resulting from laser-induced hyperthermia alone and in combination with photodynamic therapy (PDT) in the treatment of rat liver tumours by means of scanning electron microscopy. A total of 18 Wistar rats were divided into three groups. Group I (six animals) underwent hyperthermia for 15 min (15-min hyperthermia). Group II (six animals) underwent hyperthermia for 30 min (30-min hyperthermia). Group III (six animals) received the combined treatment of PDT and 30-min hyperthermia. For PDT, delta-amino laevulinic acid at a dose of 60 mg/kg of body weight was intravenously administered 60 min before irradiation at 635 nm. The morphological results indicated that 15-min hyperthermia gave rise to an increase in permeability of the vessels in the treated tumour. Thirty-min hyperthermia caused extreme oedema of vascular endothelial cells and restrictive openings of tumour branch vessels. The combined therapy of PDT and hyperthermia destroyed tumour vasculature. Large breaks of the inner wall of the treated tumour vessels were deeply involved in the basement membrane of the vessel. The results indicate that there may be a close link between inhibition of tumour growth and degree of damage to tumour vessels.

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.

Laser Doppler perfusion imaging: New technique for determination of perfusion and reperfusion of splanchnic organs and tumor tissue

Several investigations indicated that laser Doppler flowmetry on the liver surface reflects relative changes of the total liver blood flow. In this study, Laser Doppler Perfusion Imaging (LDI), monitoring the surface only, was used for measurements of tissue perfusion of normal and/or impaired liver, pancreas, spleen, stomach and intestine, and the blood flow of hepatic tumors in rats.

Immunotherapy in liver tumors: II. Intratumoral injection with activated tumor-infiltrating lymphocytes, intrasplenic administration of recombinant interleukin-2 and interferon α causes tumor regression and lysis

This study tested the effect of intratumoral injection with activated tumor-infiltrating lymphocytes (TIL), and simultaneous administration of recombinant interleukin-2 (rIL-2) and interferon alpha (IFN-alpha) (LII protocol), on mouse liver tumor. Group I (n = 10) served as the controls. Group II (n = 17) received rIL-2 + IFN-alpha schedule. Group III (n = 20) received the LII protocol. A total of 5 x 10(6) of TIL were injected into 4 sites of a tumor in a single treatment. rIL-2 (1 x 10(6) IU) on the first day and IFN-alpha (1 x 10(5) IU) on the second day were alternately given with a total of 10 treatment doses that were completed in 20 days. Tumor remission or regression rates of 29% and 40% were obtained in groups II and III, respectively, but no remission was obtained in the controls. A large number of TIL were also observed in the tumors treated with the LII protocol. Making comparisons between the control group and IL-2 + IFN-alpha schedule, and the control group and LII protocol, the ratios of cytolytic activity of TIL in vitro were 0:32 and 0:57, respectively. We conclude that the LII protocol appears to be more effective in the treatment of mouse liver tumor than the IL-2 + IFN-alpha schedule, and that it may be a new promise for the treatment of patients with liver malignancies.

Intra-operative laser-induced photodynamic therapy in the treatment of experimental hepatic tumours

Objective: To examine the effect of photodynamic therapy (PDT) on experimental liver tumours in rats. Design: An experimental liver tumour model was used. Each of a group of rats had two turnours simultaneously inoculated into its liver. The tumour located in the left hepatic lobe was used for PDT, and the other one, in the median lobe, as a control. The haem precursor &dgr;-amino laevulinic acid (ALA), at a dose of 30 mg/kg body weight, was injected 60 min before laser irradiation. Rats in group I received ALA through a femoral vein. Those in group II received ALA through the portal vein. Group III had an injection of ALA solution through the portal vein plus hepatic inflow occlusion. Three and 6 days after the treatment, the rats were killed, and the tumours were measured, and ultrastructural changes were examined using scanning electron microscopy. Setting: Lund University Medical Laser Centre, Lund, Sweden. Results: The mean tumour volume of the treated tumours increased by factors of 1.9, 1.5 and 1.7 in groups I, II and III, respectively, compared with the pretreatment baseline value. However, the mean tumour volume in the control tumours increased by factors of 9.5, 4.3 and 4.8 in the respective groups. Under the light microscope, marked necrosis of the treated tumour and the surrounding liver tissue was observed. Scanning electron microscopy revealed heavy damage to the cells and vessels in the treated tumour. Conclusion: PDT with ALA is an effective treatment modality for rat liver tumours.

Immunotherapy in liver tumors: III. A new experimental model of metastatic liver tumors from colorectal carcinoma for cytokine therapy

A new model of metastatic liver tumors in Wistar/Furth rats is introduced. A colorectal adenocarcinoma cell line (LDLX40) induced by 1,2-dimethylhydrazine was injected through one of the branches of the ileal mesenteric vein to develop metastatic liver tumors in rats. On day 30 after the inoculation of tumor cells, micrometastases were detected under microscopy in all animals that received tumor inoculation. Macrometastases in 87.7% of animals were found by either the tumor staining test or ultrasonography. No extrahepatic tumor developed in this tumor model. To observe the effects of different treatment strategies on metastatic liver tumors, 35 animals were randomly divided into four groups. Group I served as control. Group II underwent hepatic artery ligation (HAL). Group III received intraportal administration of recombinant interleukin-2 (rIL-2) and interferon-alpha (IFN-alpha). Group IV had intraportal medication of rIL-2 and IFN-alpha + HAL (the IIH protocol). Results indicated that rapid tumor growth was seen in the control tumors. HAL produced little response to metastatic liver tumors as compared to the control group (P > 0.05). The combined application of rIL-2 and IFN-alpha showed an improved result, with 22% of tumor growth inhibition or regression (P < 0.05 compared to the control group). Twenty-eight percent of tumor growth restraint or regression was found in the group treated with the IIH protocol (P < 0.05 compared to the control group). We conclude that this new experimental model of metastatic liver tumors is reproducible, and that the IIH protocol is effective in the treatment of metastatic liver tumors in rats. These beneficial effects from the IIH protocol may be introduced into patients with metastatic liver tumors.

Immunotherapy in liver tumours: I. Combined administration of recombinant interleukin-2 (IL-2) and interferon-α through the subcutaneous transposed spleen prolongs the half-life of IL-2 in vivo and enhances antitumor effects

Two modifications in this study, including the use of subcutaneous transposed spleen (STS) as a port for administration of recombinant interleukin-2 (IL-2) and interferon-alpha (IFN-alpha), and the mixture of IL-2 and IFN-alpha with degradable starch microspheres (IIM), for the treatment of rat liver tumor are introduced. Group I is the control. Group II received the IIM schedule through the STS. Group III and group IV received IL-2 and IFN-alpha, diluted with normal saline and injected through the STS or a peripheral vein. The comparative studies indicated that the best result was seen in group II where the elevated concentration of IL-2 in portal blood and massive tumor necrosis with lysis were observed. Inhibitions of tumor growth of 33%, 20% and 13% in group II, III, and IV, respectively, were observed. We conclude that administration of the IIM schedule through the STS is an effective method for the treatment of liver tumor in rats.

Repeated immunotherapy using intratumoural injection with recombinant interleukin-2 and tumour-infiltrating lymphocytes inhibits growth of breast cancer and induces apoptosis of tumour cells

This study tested the effect of repeated intratumoural injection with recombinant interleukin-2 (rIL-2) and tumour-infiltrating lymphocytes (TILs) on inhibition of growth of breast cancer and on induction of apoptosis of tumour cells. The tumour cell line LDLX43 was used to induce breast cancer in Wistar rats. Group I (10 rats) was the control. Group II (12 rats) received repeated intratumoural injection with rIL-2 and TILs. rIL-2 at the dose of 5 x 10(5) IU/day was given for 7 days, and 1 x 10(7) TILs were injected on the second day of each rIL-2 therapy, for a treatment session. Overall, two treatment sessions of immunotherapy with rIL-2 and TILs were given in all treated animals. Rapid increased tumour volume was found in the control group. In the treated group the total response rate was 42%, of which 25% tumours showed partial regression and 17% tumours reached complete remission where infiltration of plenty of T lymphocytes was detected, indicating that T cell-mediated antitumour immunity is primarily responsible for tumour rejection. Further investigation showed the repeated immunotherapy using intratumoural injection with rIL-2 and TILs could induce the development of apoptosis of breast cancer cells.

Beneficial-effects of Platelet-activating-factor Receptor Antagonist Web-2170 On 90-minute Hepatic Inflow Interruption

Objective: To study the effects of different doses of platelet activating factor receptor antagonist WEB 2170 on animal survival, haemodynamics, reperfusion and ultrastructural changes in the ischaemic liver in rats undergoing 90-min total hepatic inflow interruption (THII). Design: Sixty-five rats were divided into five groups. All animals underwent 90-min THII. Group 1 served as controls. Group 2 underwent THII alone. Group 3 received an intravenous injection of 1 mg/kg WEB 2170 prior to THII. Group 4 received a bolus injection of 3 mg/kg WEB 2170 before THII. Group 5 received 3 mg/kg WEB 2170 before, during and after THII. The liver reperfusion index using laser Doppler flowmetry, the time of ischaemic liver initiative reperfusion, and scanning electron microscopy were performed to evaluate the results of different dose schedules. Setting: Lund University Hospital, Lund, Sweden. Results: Animal survival rate, liver reperfusion index, the time of ischaemic liver initiative reperfusion and ultrastructural damage of the ischaemic liver were markedly improved in the groups treated with WEB 2170 compared with the non-treated 90-min THII group. The best result was obtained in the group receiving the three separate doses. Conclusion: In the 90-min THII model, WEB 2170 protects the liver from ischaemia-reperfusion injury and the spread of damage to the post-stasis splanchnic organs. These beneficial effects may be extended to hepatic transplantation or major resections of the liver.