The cells were then inoculated under standard conditions into mice, and the growth of ascites tumour was determined.Ehrlich ascites cells or another strain derived from a spontaneous mouse mammary carcinoma (DiVita's ascites cells) were incubated vitro at pH 7.4 for 30 minutes. This retardation of tumour growth was not dependent on the presence of glucose in the incubation medium and could be observed in the presence of about 5% ascitic fluid. However, the retardation appeared to be considerably less marked (though readily detectable) when the incubation with Synkavit was performed anaerobically.On this basis, Synkavit has been shown to retard the growth of ascites tumour provided that the cells were incubated in The retardation of tumour growth by Synkavit was abolished completely by simultaneous incubation with excess ATP or partially by equimolar ATP. Simultaneous incubation with excess ADP also abolished the retardation by Synkavit of the growth of tumour. Moreover, ATP addition to the medium at a later period appeared to be partially successful in abolishing the Synkavit effect on tumour growth.The mechanism by which ATP reduced the growth-inhibitory effect of Synkavit has been partially clarified by investigating the effect of ATP on the incorporation of a tritiated derivative of Synkavit, TRK 219. The results show that in Ehrlich ascites cells, simultaneous incubation in Spinner-medium with excess, or equimolar, ATP reduced the incorporation of labelled metabolites of Synkavit by 78% or 14% respectively. On the other hand, in a continuous cell line of human epithelial cells (HEp/2), excess ATP reduced the incorporation of metabolites of labelled Synkavit very slightly.These results have been discussed in the light of other evidence to consider the mechanism whereby ATP reduced the growth-inhibitory effects of Synkavit.


P < 0.005) and multivariate analysis (P = 0.02). p53, bcl-2, steroid hormone receptor and c-erbB2 immunostaining were scarcely affected. Baseline bcl2 (P = 0.04) and c-erbB2 (P = 0.02) were directly and inversely associated with the reduction in Ki67 immunostaining, respectively. Baseline p53 expression (P < 0.01) was directly related with Ki67 expression at residual tumour, whereas oestrogen receptor expression (P < 0.001) was inversely related. Ki67 at residual tumour was a better predictor for relapse-free survival (RFS) than baseline Ki67. Clinical response (P < 0.03), but not reduction in Ki67, was a significant independent predictor for disease recurrence. Chemotherapy was found to induce tumour shrinkage and to reduce the number of cells in the cell cycle, but its effect on tumour biology/aggressiveness was minimal. Reduction in Ki67 immunostaining correlated with clinical response but failed to be related to RFS. Ki67 expression at surgery rather than at baseline appears to be a better predictor for disease relapse. © 2001 Cancer Research Campaign  http://www.bjcancer.comThe association between tumour shrinkage and reduction in kinetic cell activity after primary chemotherapy in human breast cancer is still a matter of investigation. 157 patients with T2-4, N0-1, M0 breast cancer received primary chemotherapy consisting of either the CMF regimen + tamoxifen (the first consecutive 76 cases) or the single agent epirubicin (the subsequent 81). Ki67, p53, bcl2, c-erbB2 and steroid hormone receptors were evaluated immunohistochemically in tumour specimens obtained before chemotherapy and at surgery. Tumour shrinkage of >50% occurred in 72.4% of patients. Ki67 expression significantly decreased after chemotherapy; the reduction correlated with tumour response in both univariate (


The chick embryo was used to study the relationship between the onset of tumour neovascularization and tumour growth. Walker 256 carcinosarcoma was implanted on the chrioallantoic membrane (CAM) of about 600 embryos aged 5-16 days. Tumour diameter and changes in the CAM vasculature in response to the implants were recorded daily. Representative tumours were examined by light microscopy of Epon-embedded tissue and autoradiography after injection of [3H]-thymidine. Tumours remained avascular for 72 h, after which they were penetrated by new blood vessels and began a phase of rapid growth. The rate of growth during this vascular phase was greatest for implants on 5- and 6-day-old embryos and decreased the later the day of implantation. The time of onset of tumour angiogenesis appears to be independent of the immunological state of the chick embryo, although the rate of growth after vascularization may be modified by the onset of immunity. This study suggests that the avascular and vascular phases of tumour growth are separable, and that the avascular tumour population lives under the growth constraints which limit the size of a tumour spheroid growing in soft agar or aqueous humour.


Corynebacterium parvum was labelled by growing live bacteria in the presence of [3H]thymidine. The bacteria were killed by formalin, washed thoroughly and resuspended at a concentration of 7 mg dry weight/ml. An activity of 1-6 X 10(5) ct/min/0-1 ml was obtained. The biological properties  of the labelled C. parvum were compared with those of commercially available vaccine, and were found to be similar. Labelled C. parvum was injected i.v., i.p., or s.c. into normal C57BL mice and the localization of activity determined at 4 h and 1,3,7 and 14 days after injection. After i.v. or i.p. injection, highest counts were recorded in the liver. Moderate activity was found in the spleen, lungs and small gut. After s.c. injection, the majority of radioactive label was detected at the site of injection and little found in other tissues. The distribution of injected C. parvum was also studied in mice bearing Lewis tumour, and was found to be similar to that in normal mice. Moderate amounts of labelled C. parvum were recovered from tumour. There appeared to be no relationship between the antitumour effect of C. parvum given by a particular route of injection and the concentration of C. parvum recovered from the tumour.


Tumour metastasis in BCG-pretreated mice was studied using a methylcholanthrene-induced fibrosarcoma in C3H/He mice. When tumour cells were injected into the BCG-primed site, distant metastasis occurred in the lungs and the popliteal lymph node, through this tumour did not metastasize in normal mice. Such metastases were increased in proportion to the number of tumour cells injected into the BCG-primed site, and developed soon after tumour challenge. Concomitant immunity developed well in the mice bearing such metastases, but did not inhibit metastatic growth. Experiments using 125I-labelled SRBC or tumour cells revealed that such cells egressed rapidly from the BCG-primed site. When the tumour was inoculated into the contralateral foot to the BCG-primed site, the incidence and the number of metastases was reduced. Furthermore, BCG infection induced an increase of platelet count. I.v. injection of this tumour induced marked thrombocytopenia in normal mice. Administration of pentoxifylline, a methylxanthine derivative before tumour challenge reduced such metastases. These findings suggest that the changes in peripheral blood, such as increased platelet count and increased release of tumour cells from the injection site, facilitated distant metastasis in BCG-pretreated mice.


Surgeons have long been concerned that cancer may be disseminated by shedding of tumour cells into the bloodstream during surgery. Early claims that cancer operations induced an increase in the number of tumour cells shed into the circulation were subsequently discredited, and the issue has remained unresolved. We used immunocytochemistry for cytokeratins to detect tumour cells in effluent blood from breast carcinomas in 18 patients undergoing surgery. Tumour cells were detectable in 6/18 patients during surgery, in only one patient before operation and in none post-operatively (P = 0.025). Circulating cells were associated with vascular invasion within the primary tumour (P = 0.032). No cytokeratin-positive cells were found in blood from ten normal volunteers or four patients undergoing surgery for other breast conditions. These results confirm that cancer surgery in humans results in an increase in the shedding of tumour cells into the circulation. The implications for prognosis and practice should be determined by larger prospective studies.


Several injection schedules were also employed. On the whole, reasonable immunoprotection was obtained. The results, when allogeneic hybrid or xenogeneic cells were used, were comparable with those obtained for polyoma virus. This indicates that the T.A.T.A. of the SEWA tumour had been well conserved. When polyoma virus alone was used as the protector agent, injection 7 days before the tumour challenge, or iterative injection, afforded better protection than a single injection on the day of challenge.Various procedures for immunoprotection in the A-SW mouse SEWA tumour system were compared. Four types of protector agents were used: polyoma virus, allogeneic S724 cells, A9/SEWA hybrid cells and xenogeneic ST


Previous studies suggested that aprotinin might enhance the host's immunological resistance to tumours. This possibility has now been further investigated by studying the behaviour of tumours in both hamsters and mice. A second tumour graft in tumour-bearing hamsters appeared more rapidly than the first. Prior administration of aprotinin abolished this effect. Pretreatment of non-cancerous mice with cortisone nullified the effectiveness of aprotinin in inhibiting the growth of a subsequent tumour graft. These results are interpreted as additional evidence that aprotinin enhances the immunological system against tumour cells.


Tumour microvascular density (MVD) was assessed by manual and computerized morphometrical analysis. Expression of vascular endothelial growth factor (VEGF) was studied by in situ hybridization. Radiotherapy decreased MVD to 157 vessels per mm2 compared to 217 vessels per mm2 in controls. Estramustine counteracted this anti-angiogenic effect and potentiated the anti-tumoural effect of radiotherapy. In addition, vessel size increased after estramustine treatment. Five days after completion of radiotherapy the expression of VEGF was increased in the centre of the tumours. In conclusion, fractionated radiotherapy decreases microvascular density in experimental malignant glioma. This effect was abolished by estramustine. The anti-vascular effect of irradiation is important to recognize when combining radiotherapy with cytotoxic drugs. © 1999 Cancer Research CampaignTumour angiogenesis is essential for progression of solid tumours and constitutes an interesting target for therapy. However, impaired tumour blood supply may also be an important obstacle for treatment by radiotherapy and chemotherapy. Estramustine has been shown to increase tumour blood flow and potentiate the effect of radiotherapy in experimental glioma. This study investigated the effects of fractionated radiotherapy and estramustine on angiogenesis in malignant glioma. The intracerebral BT4C rat glioma model was used and the animals were given whole brain radiotherapy 4 Gy × 5 days alone or in combination with estramustine 20 mg kg


These results might in part explain the observed difference in metabolism of chlorambucil by the resistant and sensitive strain of Yoshida ascites sarcoma cells.Drug uptake into the body tissue was rapid, with a high level of radioactivity being associated with the plasma and ascitic fluid during the initial 6-hour period after treatment. Previous studies


The relationship between carcinoembryonic antigen (CEA) and A antigenic determinants on the cell surface of colon-tumour cells was studied by the ADCC assay. Antiserum prepared in 2 rabbits to an undecapeptide analogous to the amino terminal of CEA(1-11) was found by us either to participate in (Rabbit 2) or specifically inhibit (Rabbit 1) ADCC. The binding spectra of these two antisera and of antiserum to the whole CEA molecule were similar. All of them react with A and non-A colon-tumour cells as well as red blood cells of Type A (RBC-A) and their activity was completely absorbed on RBC-A but not on B or O. O-type, ADCC-reactive human sera always react with A-type colon-tumour cells and RBC-A, and some of them with non-A colon-tumour cells also. The degree of inhibition of their reactivity by anti-CEA(1-11) R1 varied between sera, from none to almost a complete inhibition, and is not related to whether the serum is of cancer or non-cancer origin. Non-reactive O-type sera contain anti-A activity demonstrable by haemagglutination and immunofluorescence. However, they cannot participate in ADCC reaction nor inhibit it. The sera, which contain lymphocyte-dependent antibody to A-type colon-tumour cells, lysed RBC-A, without the addition of lymphocytes or complement, in an immunologically specific way. It is concluded that the reactivity seen in our ADCC system is related to a determinant common to A and CEA  which most probably resides in the amino terminal part of these molecules. This determinant elicits the production of lymphocyte-dependent antibodies in about 50% of people with blood group O. Thus, the amino terminal part of CEA is not a tumour-specific part of the CEA molecule. No specific anti-tumour activity was found in patients' serum by this method, and claims for its demonstration by other methods may well be related to the non-specific activity observed here.


I.p. administration of 1.4 mg Corynebacterium parvum (C. parvum) 24 h before inoculation of Landschütz ascites carcinoma (LAC) cells significantly impaired growth of the tumour in MF1 mice. The injection of tumour cells caused a transient inhibition of the activity of the mononuclear phagocyte system (MPS) in both normal and C. parvum-treated hosts, as evidenced by impaired clearance of colloidal carbon from the bloodstream and reduction in hepatic phagocytosis of 51Cr-labelled sheep erythrocytes. Depression in Kupffer-cell activity was associated with a shift in particle distribution towards the spleen. The pronounced hepatosplenomegaly in response to C. parvum was significantly less in animals which also received tumour cells. Histological examination of liver and spleen revealed evidence of depressed MPS activity. Granuloma production in the liver in response to C. parvum was inhibited in tumour-bearing mice, and macrophage proliferation within the spleen was also reduced. Ascitic fluid showed similar inhibitory effects to those of tumour-cell suspensions, suggesting production by LAC of a heat-stable macrophage-inhibitory factor.


The purified bacterial chondroitinase-ABC significantly inhibited the growth of tumour; chondroitinase-AC inhibited the tumour to some extent but chondro-4-sulphatase, chondro-6-sulphatase, streptomyces hyaluronidase, and β-glucuronidase had no inhibitory effect. Heat-treated chondroitinase-ABC had no inhibitory effect on the tumour growth. The growth of tumour cells which were injected subcutaneously after The injection of 1 ml of chondroitin sulphate A and chondroitin sulphate C solution prior to tumour inoculation into the same site promoted the tumour growth, while growth-stimulating effect of chondroitin sulphate B was ambiguous. The chondroitin sulphate appears to serve as a growth supporter which protects the surface of tumour cells and promotes the physiological surface function of the cells.


The reduced tumour blood flow and oxygenation correlated well with the extent of tumour necrosis and tumour cells apoptosis induced by electrochemotherapy with cisplatin. Therefore, the data indicate that antitumour effectiveness of electrochemotherapy is not only due to increased cytotoxicity of cisplatin due to electroporation of tumour cells, but also due to anti-vascular effect of electrochemotherapy, which resulted in reduced tumour blood flow and oxygenation.Electrochemotherapy is an antitumour treatment that utilises locally delivered electric pulses to increase cytotoxicity of chemotherapeutic drugs. Besides increased drug delivery, application of electric pulses affects tumour blood flow. The aim of this study was to determine tumour blood flow modifying effects of electrochemotherapy with cisplatin, its effects on tumour oxygenation and to determine their relation to antitumour effectiveness. Electrochemotherapy of SA-1 subcutaneous tumours was performed by application of electric pulses to the tumours, following administration of cisplatin. Tumour blood flow modifying effects of electrochemotherapy were determined by measurement of tumour perfusion using the Patent blue staining technique, determination of tumour blood volume, and microvascular permeability using contrast enhanced magnetic resonance imaging, and tumour oxygenation using electron paramagnetic resonance oximetry. Antitumour effectiveness was determined by tumour growth delay and the extent of tumour necrosis and apoptosis. Tumour treatment by electrochemotherapy induced 9.4 days tumour growth delay. Tumour blood flow was reduced instantaneously and persisted for several days. This reduction in tumour blood flow was reflected in reduced tumour oxygenation. The maximal reduction in partial oxygen pressure  87, 1047–1054. doi:10.1038/sj.bjc.6600606www.bjcancer.comCancer Research UK© 2002  Electroporation, i.e. application of electric pulses to the cells or tissues, induces under suitable conditions reversible permeabilization of cell membrane, thus facilitating entry into the cells of nonpermeant or poorly permeant molecules . Electroin vitro on several rodent and human cell lines  and tumour staining with Patent blue were used, as well as electron paramagnetic resonance (EPR) oximetry.2O at a concentration of 1 mg ml−1. Final concentration was prepared in Eagle's minimal essential medium  for experiments in vitro or in 0.9% NaCl solution for experiments in vivo. For each experiment, fresh solution of cisplatin was prepared.cis-Diamminedichloroplatinum (II) (cisplatin) was obtained from Bristol Myers Squibb  as a crystalline powder and dissolved in sterile Hin vitro studies, they were grown in EMEM supplemented with 10% foetal calf serum  in a humidified atmosphere at 37°C containing 5% CO2. The cells were routinely subcultured twice a week.Murine fibrosarcoma SA-1  cells were used for the experiments. For the ad libitum. Before the experiments, the mice were subjected to an adaptation period of at least 10 days. The mice were 10–14 weeks old at the beginning of the experiments, weighing 20–25 g.A/J mice, of both sexes, purchased from the Institute Rudjer Boskovic  were used for this study. They were kept at constant room temperature (22°C) with a natural day/night light cycle in a conventional animal colony with food and water 5 SA-1 cells in 0.1 ml 0.9% NaCl solution. Six to 8 days after implantation, when tumours reached approximately 40 mm3 in volume mice were randomly divided into experimental groups. Treatment protocols were approved by the Department of Agriculture of the Republic of Slovenia No. 323-02-183/99. The ethical guidelines that were followed meet the standards required by the UKCCCR guidelines  and 10% ketamine  administered i.p. During anaesthesia, body temperature was kept at physiological values by a regulated heating pad on which the mice were placed .Tumour cells were obtained from the ascitic form of the tumours in mice, serially transplanted every 7 days. Solid subcutaneous tumours, located dorsolaterally or ventrally in mice, were initiated by an injection of 5×107 cells ml−1) was mixed with 10 μl of different stock concentrations of cisplatin, as described previously  and the other half served as a control for cisplatin treatment alone. The final cisplatin concentrations ranged from 0.5 to 400 μg ml−1. The cells were incubated for 5 min after electroporation, and thereafter clonogenic assay was performed , with pulse width of 100 μs and repetition frequency 1 Hz were delivered by two flat, parallel stainless-steel electrodes 8 mm apart , which were placed percutaneously at the opposite margins of the tumour. Good contact between the electrodes and the skin was assured by means of conductive gel. Electric pulses were generated by an electroporator Jouan GHT 1287 . In the electrochemotherapy protocol, mice were treated with electric pulses 3 min after cisplatin injection. All treatments were performed without anaesthesia and were well tolerated by the animals  with a vernier caliper three times per week. Tumour volumes were calculated by the formula V=π×e1×e2×e3 /6. The arithmetic mean of tumour volumes and standard error of the mean (s.e.) were calculated for each experimental group. Tumour doubling time was determined for each individual tumour from the growth curves. Tumour growth delay was calculated for each individual tumour by subtracting the doubling time of each tumour from the mean doubling time of the control group and then averaged for each experimental group.Tumour growth was followed by measuring three mutually orthogonal tumour diameters (e86RbCl extraction technique) measuring relative tumour blood flow, good correlation between both methods was found (r=0.962) (86RbCl extraction technique gives measure of tissue perfusion as a function of cardiac output and is expressed as percent injected activity per gram or relative uptake (per cent control)  was used to estimate tumour perfusion. Patent blue (1.25 %) was injected (0.2 ml) at different time points after treatment into the tail vein of animals with tumours from control, electric pulses, cisplatin and electrochemotherapy groups. After the dye was distributed evenly through the tissue (1 min), animals were killed by cervical dislocation which results in instantaneous death of the animal and tumours excised, carefully removed from the skin, cut in half along their largest diameter, and immediately evaluated. The percentage of stained area for each of the faces of the two halves of tumour cross-section (perfused), as opposed to nonstained area (nonperfused) was visually estimated by three individuals in blind fashion. The mean of both estimations was used as an indicator of tumour perfusion. Because of marked differences in tumour staining between well-perfused and poorly perfused tumours, visual estimation of the whole tumour cross-sections gives good estimation and reliable results. In the study comparing Patent blue staining and the pharmacological method (control) .−1 in a bolus via 23-gauge intravenous cannula inserted into a tail vein of anaesthetised mice. No systemic toxicity was observed. Animals were placed in the 2.35 T Bruker Biospec system with a horizontal bore magnet.An intermediate molecular size macromolecular magnetic resonance contrast agent gadomer-17  was used to assess changes in fractional tumour blood volume (BV) and microvascular permeability surface area product (PS) in control, electric pulses, cisplatin and electrochemotherapy treated animals. When administered i.v., this agent does not diffuse through endothelium, unless there is a pathological change  with dimensions 32×256 k space data points was acquired repetitively for 70 min (80–100 ‘key’ images). Each key image was acquired with 32 phase-encoding steps that took 38 s. Before the reconstruction, the subset was first completed in remaining k-space points  with the data from the pre-contrast acquisition. Finally, images were reconstructed with two-dimensional inverse Fourier transformation.A pre-contrast image (complete k-space data set) was acquired first, using a standard spin-echo technique. Imaging parameters were: repetition time, TTC, of the gadomer-17 in the tumour was calculated by subtraction of the pre-contrast signal from the post-contrast signals on a pixel-by-pixel basis. The T1 relaxivity in water Gd−1 at 20 MHz and 39 °C is 14.4 mmols−1 , the magnetic resonance signal was measured within the tumour and vena cava in pre-contrast image and at least 80 post-contrast images that were acquired every 38 s. The signal was corrected for signal variations against a water phantom. From the measured signal, the concentration Hct is the measured haematocrit of the blood  and PS′ is linear fit coefficient of T/CBC at different time points. BV, which represents fraction of contrast agent leaking through tumour endothelium into interstitial space in time was calculated as:where TC is concentration of contrast agent in tumour and BC concentration of contrast agent in blood.where 2) in tumours and subcutis on the contralateral side before and after treatment of tumours with electric pulses, cisplatin and electrochemotherapy. EPR oximetry is a non-invasive method, (after insertion of the paramagnetic probe) which allows monitoring of pO2 repeatedly at the same point in the tissue over long periods of time  made from Bubinga tree , which is sensitive to oxygen was implanted into tumour center and periphery (tumour volume approximately 40 mm3) and subcutis one day before the treatments and its EPR spectral line-width measured at different time points up to two days after treatment , both designed by Professor T Walczak . Typical spectrometer settings were: modulation frequency, 100 KHz; modulation amplitude not more than one-third of the peak-to-peak line-width, and scan range, 2 mT. The line width of the EPR spectra reflects the pOThe same tumours that were used for Patent blue staining were fixed in 10% buffered neutral formalin. Sections through the largest diameter of the tumours were embedded in paraffin, cut and stained with haematoxylin and eosin by standard method. Necrosis in slides of 3–9 tumours per group were determined in blind fashion by three experts. The percentage of necrotic area was determined in whole mount tumour sections.The apoptotic rate was determined using Cell Death Detection ELISA . The assay is based on the quantitative sandwich-enzyme-immunoassay principle and detects apoptotic cells relatively selectively even in the presence of an excess of necrotic cells  was used for statistical analysis. P levels of less than 0.05 were taken as significant.All data were tested for normality of distribution using the Kolmogrov–Smirnov test. The differences between the mean values of groups were tested for significance by a in vitro by a colony forming assay. Exposure of cells to electric pulses resulted in a marked increase in cisplatin cytotoxicity. Throughout the range of cisplatin concentrations investigated, cells exposed to electric pulses were more sensitive to cisplatin than those that were not  gradually increased over 70 min observation time . The tumour enhancement increased gradually, indicating diffusion of the contrast agent from the blood into the interstitial space. Tumours treated with electrochemotherapy showed delayed enhancement with gadomer-17, indicating a change in blood flow or decreased permeability. The concentration of contrast agent in the tumour  were higher than in the untreated tumour tissue  than in the tumour centre (5.3 mmHg).EPR oximetry was used to measure pO2 changes in tumours was similar to the changes in tumour perfusion, as measured by Patent blue staining. A very fast and profound reduction of pO2 was observed in the tumours after application of electric pulses to the tumours and after electrochemotherapy. The maximal reduction was observed within 2 h after the treatment, with steady recovery thereafter. The recovery to pretreatment levels was slower after electrochemotherapy (48 h) than after application of electric pulses (8 h). Treatment with cisplatin only and effects on subcutis, contralateral to the application of electric pulses or electrochemotherapy had only short lasting effects on pO2 .The time course of pOThe time course of tumour necrosis was evaluated for 5 days after treatment. The extent of necrosis in the cisplatin treated tumours was similar to the untreated tumours. Application of electric pulses to the tumours induced a slow increase in tumour necrosis up to 8 h after treatment, and remained on this level up to the 5th day. In electrochemotherapy treated tumours the degree of tumour necrosis followed the onset of tumour necrosis in the group treated with electric pulses only, and continued to increase up to the 3rd day after the treatment, when it reached 90% and it remained at this level through the 5th day  and application of electric pulses (0.8 days tumour growth delay). The effect was less pronounced than in electrochemotherapy with bleomycin, which is consistent with in vitro data on endothelial cells sensitivity (Reduced tumour blood flow is expected to result in lower oxygen tension. By using EPR oximetry we were able to follow the time course of oxygen tension in the tumours. Electrochemotherapy induced profound and long lasting pOThe proportion of apoptotic cells increased only after electrochemotherapy with cisplatin. Cisplatin is known to induce apoptotic cell death, therefore it can be speculated that electrochemotherapy, which increases cisplatin delivery into the cells, also stimulates apoptotic cell death . In histIn conclusion, this study provides data on physiological changes in the tumours, as a consequence of electrochemotherapy with cisplatin and correlates these effects with antitumour effectiveness and histological changes in the tumours. The results indicate that reduction in tumour blood flow and tumour oxygenation correlate with extensive tumour necrosis and induction of apoptosis of the tumours treated by electrochemotherapy with cisplatin. The data support the evidence that the antitumour effectiveness of electrochemotherapy is not only due to increased drug delivery to the cells, but also to anti-vascular effect of electrochemotherapy, which results in reduced tumour blood flow and oxygenation.


The effect of the O6-alkylguanine-DNA alkyltransferase (AGT) inhibitor, O6-benzylguanine (O6-BG), on the anti-tumour activity of 8-carbamoyl-3-methylimidazo -1,2,3,5-tetrazine-4(3H)-one (temozolomide) or 1,3-bis(2-chloroethyl)-nitrosourea (BCNU) was evaluated in athymic mice bearing subcutaneous (s.c.) human glioma (U87MG) xenografts. The activity of AGT in U87MG xenografts was 4.3 +/- 1.5 fmol mg-1 protein (mean +/- s.d). These xenografts were inherently sensitive to treatment with alkylating compounds alone, with non-toxic doses of temozolomide (35 mg kg-1) or BCNU (10 mg kg-1) producing tumour growth delays of 23.3 and 11.8 days respectively. O6-BG (40 mg kg-1) did not inhibit tumour growth when administered alone, but was found to enhance significantly the anti-tumour activity of temozolomide or BCNU when administered 1 h before therapy . AGT activity measured 24 h after the administration of 40 mg kg-1 O6-BG, was only 0.9 +/- 0.2 fmol mg-1 protein. These results are in contrast to previous studies in vitro with tumour cell lines of low AGT activity (< 15 fmol mg-1 protein), in which the cytotoxicity of temozolomide or BCNU was unaffected by AGT depletion.


Glucose and lipid metabolism in the brain, liver and in a transplanted tumour were found to be variously altered within 2 to 3 h of administering single doses of the radiosensitizer Ro-03-8799 to normal and tumour-bearing mice. Hepatic lactate and glycerol-3-phosphate (G3P) levels were decreased but those of the ketone body beta-hydroxybutyrate (beta-HOBu) were raised. However, in the tumour, these levels were all enhanced. The lactate levels in brain remained relatively constant but both beta-HOBu and G3P levels were altered in a manner similar to that in the liver. The levels of glucose were approximately doubled in blood, brain and tumour, but whereas tumour G6P levels increased, those in the brain were lowered to below the limits of detection. Hepatic glucose levels were significantly decreased after 1 h but G6P levels were not affected. These changes could neither be related to inhibitory effects on hepatic glucokinase or brain hexokinase activity nor to limiting amounts of ATP in both tissues. However, the activity of glucose-6-phosphatase (G6P'ase) was distinctly raised in the liver and the hepatic glycogen stores were also rapidly lowered. Overall, the results suggest that Ro-03-8799 exerts a stimulatory effect on glucose production in the liver. In both liver and brain the levels of free fatty acids and phospholipids were increased whereas those of esterified fatty acids were lowered. Most importantly, the changes in metabolite levels affect the cellular redox couples; those of the cytosol ) in both tissues is shifted towards the reduced state. These metabolic changes may result in an increase in the degree of hypoxia in the tumour and may well play an important role in the development of neuropathies.


Six patients with a total of 34 assessible subcutaneous or cutaneous lesions were treated with photodynamic therapy using 1.0, 1.5 or 2.0 mg kg-1 of photofrin II and 25-100 J cm-2 of red light (630 nm). The incidence of complete tumour response and skin necrosis were used to try to assess the therapeutic ratio of photodynamic therapy. The tumour response rate was 47%. The rate of tumour control and necrosis increased in parallel with dose of photosensitizer and light used, implying a low therapeutic ratio. However, the use of necrosis with eschar formation as an end-point for severe normal tissue damage is questioned as the skin healed completely in all cases and with minimal discomfort to the patients.


The ability of s.c. injected tumour cells to specifically inhibit the growth of similar cells injected i.v. 2 days later has been confirmed. The capacity of tumour cells to elicit this effect varies form tumour to tumour. Furthermore, it is more readily achieved with cultured than with freshly excised tumour cells. The superior effect elicited by cultured tumour cells was not overcome by treating them with trypsin or pronase. The protection achieved was impaired in T-cell-depleted mice and mice which had been irradiated (400 rad) prior to pretreatment. In contrast, it was not affected by administration of silica, sodium aurothiomolate or cortisone acetate. The results imply that T-cell-dependent responses are involved in the protection conferred by pre-injecting tumour cells shortly before i.v. challenge.


The sites of tumour development for 6 rat tumours injected into syngeneic rats via different vascular routes was determined. Xenografts of human tumours were also injected intra-arterially (i.a.) into immunosuppressed rats. Following intravenous (i.v.) and intraportal (i.ptl.) injection of cells tumour colonies localized in lung and liver respectively due to tumour cell arrest. Arterially injected radiolabelled cells disseminated and arrested in a similar distribution to cardiac output and did not 'home' to any organs. Following arterial injection of unlabelled tumour cells colonies grew in many organs. While the pattern of growth for a particular tumour varied with the cell dose, the 'arterial patterns' for all of the tumours studied followed a similar pattern. Some organs  were consistently preferred, others  only supported tumour growth following the delivery of large numbers of cells, while in some tissues (eg spleen and intestines) tumour never grew. Viable tumour cells could be demonstrated by bioassay in many organs for up to 24h after i.a. injection. However tumour growth only occurred in certain organs and the pattern of this growth was not related to the number of tumour cells arrested or their rate of autolysis. This site preference could be expressed quantitatively as the probability of an arrested cell developing into a tumour and was considered a 'soil effect'. Site preference was not directly related to organ vascularity. Organ colonisation was promoted by steroid treatment but the mechanism was unclear and was not secondary to T-cell immunosuppression or prostaglandin synthesis suppression. The adrenal glands were preferred sites of tumour growth but pharmacological manipulation of adrenal function did not alter tumour growth to this organ. Sites of injury and healing were preferred sites of tumour colonisation and this could not be accounted for by increased delivery of tumour cells to these regions. The possibility that the macrophage component of the inflammatory response promoted tumour growth was suggested from studies in which the interval between trauma and inoculation of tumour cells was varied as well as by promotion of intraperitoneal (i.p.) tumour growth by a macrophage infiltrate.


In 19 out of 27 lesions studied, we found expression of MHC II by the tumour cells, while only in one out of 11 tumour biopsies obtained from untreated metastatic melanoma patients, MHC II expression was detected. Expression of B7.1 and B7.2 by tumour cells was found in nine out of 24 and 19 out of 24 lesions, respectively. In all cases where B7.1 expression was found, expression of B7.2 by the tumour cells was also seen. In general, no or only few inflammatory cells positive for B7 were found. Expression of MHC II by tumour cells was positively correlated with the presence of tumour-infiltrating lymphocytes, regression of the lesion, and with time to progression (TTP) and overall survival (OS) of the patient. However, no significant correlation between B7.1 or B7.2 expression and regression of the tumour, TTP or OS was found. In light of other recent findings, these data altogether do support a role as biomarker for MHC II expression by tumour cells; however, its exact immunological pathomechanism(s) remain to be established.A large number of studies have indicated that specific immune reactivity plays a crucial role in the control of malignant melanoma. In this context, expression of MHC I, MHC II and B7 molecules by melanoma cells is seen as relevant for the immune response against the tumour. For a better understanding of the biological relevance of MHC II and B7 expression by tumour cells in metastatic melanoma, we studied the expression of these molecules in melanoma metastases in relation to the inflammatory response, regression of the tumour and survival from 27 patients treated with biochemotherapy (30 mg m Many studies have indicated that the immune system is capable of controlling tumour growth in cancer patients, especially in melanoma patients. Supportive findings comprise the occurrence of spontaneous regressions in primary melanomas . From stγ induces MHC class II expression in melanoma cells as well as in melanocytes . Data on the relevance of MHC II expression by tumour cells for immunological control of tumour growth is, in contrast to data on MHC I, not consistent. Melanocytes in normal skin and common nevi are negative for MHC class II  with metastatic malignant melanoma, 15 with regional disease and 12 with resectable systemic disease. The median age was 58 (range 34–71) years and their Karnofsky status was 70 or more. Recurrences were cytologically verified by fine-needle aspirates before treatment was started. One metastasis from each patient was studied. In patients with systemic disease, easily resectable metastases were chosen. In patients with regional disease, non-necrotic biopsies were randomly chosen by the pathologist. In addition, tumour biopsies obtained from 11 untreated metastatic melanoma patients  were studied for MHC II expression. All these patients had regional disease and a median age of 57 (range 39–79) years. Recurrences were cytologically verified by fine-needle aspirates. One metastasis from each patient was studied.The study was approved by the ethical committee at the University Hospital of Linköping, Sweden.−2 cisplatin (cis-diamine-dichloro-platinum) and 250 mg m−2 dacarbazine  on days 1–3 i.v., and 107 IU IFN-α2b 3 days a week s.c. The duration of one cycle was 28 days.Pretreatment investigations were as described previously  supplemented with 5.4 g l−1 of glucose for 5 min and then washed three times in Hanks' balanced salt solution , supplemented with 0.01 M Hepes solution before staining. For detection of the expression of MHC antigens, B7.1, B7.2 and the presence of T-cell subsets, the following monoclonal antibodies were used: anti-HLA, Stockholm, Sweden, DR for MHC II , anti-HLA-ABC for MHC I , anti-B7.1 , anti-B7.2 , anti-CD3 , leu-3a for CD4  and Leu-2a for CD8 . Sections for T-cell subsets were blocked with normal rabbit serum before the first staining. Sections for MHC I and MHC II were blocked with normal goat serum and sections for B7.1 and B7.2 with 10% AB serum. Tissue sections were then incubated with primary antibodies for 30 min or overnight (B7.1 and B7.2) at room temperature. Mouse IgG1  was used as a negative control for the subsets of inflammatory cells and the B7 antibodies. Mouse IgG  was used as a negative control for the major histocompatibility antigens. After the slides were washed in BSS-saponin, biotinylated rabbit-anti-mouse immunoglobulin was added at a 1 : 100 dilution in BSS-saponin for anti-CD3, anti-CD4 and anti-CD8. They were then incubated with peroxidase-labelled streptavidin  at a 1 : 100 dilution in BSS-saponin for 30 min. DAB  was used as a substrate. Sections stained for MHC I and MHC II were washed in BSS-saponin; then, goat-anti-mouse immunoglobulin was added at a 1 : 25 dilution in BSS-saponin and left to incubate for 30 min in a moist chamber. The slides were again washed in BSS-saponin, and mouse APAAP complex diluted at 1 : 25 in BSS-saponin was added and incubated for 30 min. For B7.1 and B7.2, the Envision system was used as the second step . All antibody solutions contained 2% normal blood donor AB serum. After washes in BSS-saponin and TBS, the Fast-red substrate supplemented with levamisole to block endogenous alkaline phosphatase was added to the sections (MHC and B7) and left to incubate for 20 min at room temperature. The slides were then washed in TBS, pH 7.6, counterstained in Mayer's haematoxylin for 15 min and mounted in Glycergel .Biopsies from the resected metastases were immediately snap frozen and stored at −70°C until further use. Tissue sections, 6–7 The occurrence of tumour regression was evaluated by histopath-ological examination of tumour biopsies based on the description of regressive changes in malignant melanoma as previously described . The folSurvival curves were plotted using the Kaplan–Meier method. Differences in clinical response in terms of time to progression and OS between patients showing differences in expression of MHC II or B7 by the tumour or differences in regressive changes in the tumour were analysed using the log rank test. A two-sided Fisher's exact test was used to analyse whether or not an increased level of MHC II expression coincided with an increased level of histological regression. Calculations of significance were performed using Graph Pad Prism version 3.02 for Windows .The expression of MHC classes I and II was studied in 27 lesions from biochemotherapy-treated patients by immunohistochemical analysis. All lesions were homogeneously positive for MHC I. In contrast, a heterogeneous expression of MHC II was found between and within lesions. In eight out of these 27 lesions, the tumour cells were negative for MHC II, while in the remaining 19 lesions, 10 to more than 75% of the tumour cells were positive for MHC II .Histological regression of tumour lesions was scored according to criteria as published before  and as sFigure 4ASimilar to MHC II, we plotted the extent of histological regression against the percentage of B7-positive tumour cells found in each lesion . There wP=0.034 and 0.043, respectively; log rank test for trend). The use of a cutoff point of 25% of the tumour cells positive for MHC II results in a significant difference in TTP and OS for the two resulting patient groups  regression of metastases . In none of the three aspirates positive staining of the tumour cells for MHC II was obtained, while in two of the three corresponding post-treatment tumour biopsies MHC II-positive tumour cells were detected . As also found by others  and HLA-DM . In thisIn conclusion, MHC II expression in metastatic melanoma is relevant as a biomarker for response and/or prognosis; its immunological significance, however, remains to be established. Further elucidation of the apparent complex machinery regulating expression of MHC II and its accessory molecules and expression of costimulatory molecules is warranted.


In several tumors the transketolase activity, controlled inter alia by enzymes of the pentose phosphate pathway which is an alternative, energy generating reaction-cascade to glycolysis, has been correlated with proliferation. The increase of thiamine-dependant transketolase enzyme reactions is induced especially through upregulated transketolase-like enzyme 1 (TKTL1)-activity; that shows TKTL1 to be a causative enzyme for tumors enhanced, anaerobic glucose degradation. We investigated TKTL1-expression in 88 human, formalin-fixed non-small cell lung cancer tissues and 24 carcinomas of the breast by immunohistochemical stainings applying a 0 to 3 staining-score system (3 = strongest expression). For means of validation we additionally stained 40 NSCLC fixed and paraffin-embedded utilizing the HOPE-technique; showing comparable results to the formalin-fixed, paraffin-embedded specimens (not shown). Potential correlations with age, sex, TNM-classification parameters and tumor grading as well as tumor transcription factor 1 (TTF1) and surfactant protein A (SPA) expression were investigated. 40.9% of the analyzed lung tumors expressed TKTL1 weakly (Score 1), 38.6% moderately (score 2) and 17.1% strongly (score 3). 3 tumors were diagnosed TKTL1-negative . All Breast cancer specimen stainings were positive and scored 1: 32%; scored 2: 36%; scored 3: 32%. Alveolar macrophages and Alveolar Epithelial Cells Type II were also found to be TKTL1-positive.None of the listed clinical parameters could be found to show a significant correlation to TKTL1 signal appearance.Although we describe the expression of TKTL1 in lung cancers, we need to state that up till now there is no scientific indication for any treatment regimens based upon these findings. The ability of tumors to degrade glucose, even in the presence of oxygen, through the anaerobic transketolase-dependant pentose phosphate pathway has recently shown TKTL1 to particularly influence total transketolase activity and cell proliferation ,3. GastrAfter lobectomy or pneumonectomy tumor tissues were immediately fixed with formalin and processed in the following over-night-procedure: formalin 1 h 30 min; following an alcohol series . Each step was followed by a 30 min. drop-off delay before the next step was started. After resection breast cancer specimens were processed in the same manner. Additionally we investigated 40 HOPE (Hepes Glutamic Acid Buffer Mediated Organic Solvent Protection Effect)-fixed NSCLC-tissue probes to validate the formalin fixation. After embedding in paraffin the blocks were shelved for 0–6 years within a tissue archive. For increased inter specimen comparability and even staining quality we utilized Tissue Microarrays (TMAs). These TMAs were produced from donor tissue blocks using an MTA1  device , cut and mounted on microscope slides . For homogeneous high-throughput staining conditions the tissues were stained automatically .The TMAs were deparaffinized and rehydrated by the following series at room temperature: 10 min. xylol incubation; 2 min. absolute alcohol (2 times); 2 min. 96% alcohol (2 times); 2 min. 90% alcohol; 2 min. 80% alcohol; 2 min. 70% alcohol; 2 min. aqua dest. (2 times). For antigen retrieval 30 min. citrate buffer cooking in was chosen. The primary antibody  was incubated for 30 min. in a 1:100 dilution. We used an enzyme-polymersystem  for sensitive detection with permanent AEC (Zytomed Systems) as chromogen. Tissue arrays were counterstained by incubation in Mayer's haemalum for 5 minutes. Negative-controls were included in each staining series under omission of primary antibody.The immunohistochemical stainings displayed elevated expression of TKTL1 in human lung cancer: 40.9% expressed TKTL1 weakly (score 1), 38.6% moderately score 2), 17.1% strongly (score 3), and 3.4% of the tumors were TKTL1-negative , observed in the close neighborhood of the NSCLC Figures  and 3E aA high variability of TKTL1-expression has been described in several tumor types. There is proven evidence that TKTL1-associated or -inducing, abnormal glucose degradation is increased in tumors -10. But The authors declare that they have no competing interests.HS and DK drafted the manuscript and analyzed the sections. DB was responsible for the surgical part and clinical data. EV and PZ were responsible for the histopathological and the pneumological aspects. TG conducted the study together with EV and PZ and was involved in drafting the manuscript. All authors have read and approved the present manuscript.


For each of a set of 11 methylcholanthrene-induced sarcomas of B10 mice, we measured the amount of H-2 antigen by absorption of a specific antiserum, and the strength of the tumour-specific transplantation antigen by a transplantation assay, to see whether they are correlated. No obvious correlation was seen. We showed that cell suspensions of tumours taken directly from the animal are contaminated by host cells which make a substantial contribution in H-2 assays. Since this contamination was lost after several passages in vitro, the amount of H-2 on tumour cells was assayed only after such passages.


The following information was missing from the Funding section: SM was supported by a Department of Science and Technology grant to UR. Field sampling was partially funded by WCS.


A chemical extraction assay and fluorescence microscopy incorporating a light-sensitive thermoelectrically cooled charge-coupled device (CCD) camera was used to study the kinetics of uptake, retention and localisation of disulphonated aluminium phthalocyanine (A1PcS2) and tetrasulphonated aluminium phthalocyanine (A1PcS4) at different time intervals after an i.p. injection at a dose of 10 mg kg-1 body weight (b.w.) in tumour and surrounding normal skin and muscle of female C3D2/F1 mice bearing CaD2 mammary carcinoma. Moreover, the photodynamic effect on the tumour and normal skin using sulphonated aluminium phthalocyanines  and Photofrin was compared with respect to dye, dye dose and time interval between dye administration and light exposure. The maximal concentrations of A1PcS2 in the tumour tissue were reached 2-24 h after injection of the dye, while the amounts of A1PcS4 peaked 1-2 h after the dye administration. A1PcS2 was simultaneously localised in the interstitium and in the neoplastic cells of the tumour, whereas A1PcS4 appeared to localise only in the stroma of the tumour. The photodynamic efficiency (light was applied 24 h after dye injection at a dose of 10 mg kg-1 b.w.) of the tumours was found to decrease in the following order: A1PcS2 > A1PcS4 > Photofrin > A1PcS1. Furthermore, photodynamic efficacy was strongly dependent upon dye doses and time intervals between dye administration and light exposure: the higher the dose, the higher the photodynamic efficiency. The most efficient photodynamic therapy (PDT) of the tumour was reached (day 20 tumour-free) when light exposure took place 2 h after injection of A1PcS2 (10 mg kg-1). A dual intratumoral localisation pattern of the dye, as found for A1PcS2, seems desirable to obtain a high photodynamic efficiency. The kinetic patterns of uptake, retention and localisation of A1PcS2 and A1PcS4 are roughly correlated with their photodynamic effect on the tumour and normal skin.


When used as single agents at their maximum tolerated dose, all three novel compounds produced a significant growth retardation of BCNU-resistant murine colon and human breast xenografts. This in vivo anti-tumour effect was potentiated by BG, but was accompanied by severe myelotoxicity as judged by spleen colony forming assays. However, while tumour resistance to BCNU was overcome using BG, this was at the expense of enhanced bone marrow, gut and liver toxicity. Therefore, although this ATase-depletion approach resulted in improved anti-tumour activity for all three 5-FU:CNU molecular combinations, the potentiated toxicities in already dose-limiting tissues indicate that these types of agents offer no therapeutic advantage over BCNU when they are used together with BG. © 1999 Cancer Research CampaignPrevious studies have demonstrated that novel molecular combinations of 5-fluorouracil (5FU) and 2-chloroethyl-1-nitrosourea (CNU) have good preclinical activity and may exert less myelotoxicity than the clinically used nitrosoureas such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). This study examined the effect of O


There is little evidence of marked seasonal variation. Angular regression suggested that observed differences in peak diagnosis with respect to menopausal status, tumour size, ER and PR status may be chance. © 2001 Cancer Research Campaign


Adriamycin and cyclophosphamide are active agents in human and experimental tumours. Using the L1210 murine leukaemia, their effectiveness alone and in combination was studied. The combination is highly synergistic in this tumour, resulting in a greater than 50% survival rate when the agents used alone at optimal doses are not curative. DNA synthesis by tumour cells is substantially inhibited and the total ascitic population much reduced. In contrast, DNA synthesis in sensitive host tissues is less disturbed. There is no major difference in the pharmacology of the agents whether given alone or in combination. In very advanced disease the combination is no better than treatment with cyclophosphamide alone. The combination of adriamycin and melphalan in L1210 leukaemia also produces superior results to those obtained using either drug alone at its optimal dosage.


A comparison has been made between the effects of daily insulin injection and a ketogenic diet on weight loss and tumour weight in an experimental model of cancer cachexia (MAC16). Weight loss associated with the MAC16 tumour was significantly reduced both by a ketogenic diet (80% MCT) and by daily insulin injections without an increase in either food or water consumption. Animals fed the 80% MCT diet had a significantly reduced tumour weight compared with controls fed a normal laboratory diet, while in animals administered 20 U insulin kg-1 day-1 the tumour weight was 50% greater than in saline infused controls. The stimulation of tumour growth by insulin was counteracted by the inclusion of 3-hydroxybutyrate in the drinking water without any alteration in the extent of weight loss. Depletion of both carcass fat and muscle dry weight in animals bearing the MAC16 tumour was reversed in animals administered either insulin or an 80% MCT diet. Animals bearing the MAC16 tumour had a reduced nitrogen balance compared with non-tumour-bearing controls, mainly due to excess urea excretion, and this was reversed towards control values in animals fed an 80% MCT diet, but not in animals administered insulin. These results suggest that a ketogenic diet is more effective than insulin administration in reversing the cachectic process and has the advantage of a concomitant reduction in tumour weight.


The co-injection of extracellular matrix components, such as Matrigel, with human tumour cells into nude mice has been reported to facilitate tumour formation and growth, but it is unknown whether such components exert similar effects on tumour progression and metastasis. Metastatic behaviour is known to be enhanced when tumour cells are implanted orthotopically, and it is inferred that full and efficient expression of this phenotype may involve some interactions with local connective tissue matrix. It was therefore decided to investigate whether manipulation of the mesenchymal environment by co-injection of extracellular matrix components, in the form of Matrigel, with human breast cancer cells into orthotopic or ectopic sites could augment their metastatic performance, as well as their growth at the site of inoculation. Standard aliquots of 10(6) cells of the polyclonal human breast carcinoma cell line MDA-MB-435, and of four clonal cell lines, two metastatic and two non-metastatic derived from it, were injected with and without Matrigel, orthotopically or subcutaneously into nude mice. The latent period of tumour formation at the inoculation site as well as final tumour size and metastatic performance at autopsy, 140 days after inoculation, were then assessed. The prevalence of metastasis of the parent, polyclonal, cell line and of its metastatic clones was increased if the cell inoculum was mixed with Matrigel. Non-metastatic clones were not induced to become metastatic by this treatment, but local tumour growth at the site of inoculation was enhanced in all experimental groups receiving Matrigel. Orthotopic inoculation acted synergistically with Matrigel to maximise both tumour growth and metastatic behaviour. The composition of the local extracellular matrix at the site of tumour growth influenced expression of the metastatic phenotype by cells which are constitutionally capable of this behaviour, but did not induce it in ones which are not. Previous reports that local tumour growth is facilitated by enrichment of the mesenchymal matrix are confirmed. The mechanisms by which such effects are exerted are worthy of study, to ascertain whether they might be subject to clinical manipulation designed to retard tumour growth and dissemination.


Treatment of the immunizing cells with glutaraldehyde at concentrations of 0.001% or greater for 30 min rendered these cells non-protective in tumour-rejection tests and no longer able to induce significant formation of specific antibody. However, tumour-specific antigens were shown to be expressed upon treated cells; they specifically bound tumour-specific antibody from syngeneic immune sera assessed in indirect membrane-immunofluorescence tests. Also, these cells specifically absorbed antibody from immune or tumour-bearer sera, as demonstrated in the indirect membrane-immunofluorescence test or a complement-dependent 51Cr-release test. Alloantigen expression was not influenced by glutaraldehyde treatment, although glutaraldehyde-treated hepatoma cells failed to induce alloantibody formation in KX/Not rats. Polyacrylamide-gel electrophoresis of treated cells, surface-labelled with 125I, indicated that extensive cross-linking of the surface protein occurred as a result of glutaraldehyde treatment. The present findings establish that although the expression of a tumour-specific antigen is necessary for the induction of immuno-protection against tumour-cell challenge, this alone is not a sufficient condition for eliciting tumour immunity.


WHT/Ht mice were transplanted s.c. with NC carcinoma, and the tumours were excised after 2 weeks. The mice were treated orally throughout the experiments with prednisolone 500 micrograms kg-1 or mepacrine 3.6 mg kg-1, starting the day after tumour transplantation or, with prednisolone, the day after tumour excision. In some experiments the mice were also treated with the cytotoxic drugs methotrexate 2 mg kg-1 and melphalan 1.4 mg kg-1. The excised tumours were weighed; some of them, and samples of serum, were extracted for prostanoids which were measured by radioimmunoassay. The chemotherapy lengthened the survival of the mice, but prednisolone or mepacrine had little or no effect on survival, metastasis, the response to chemotherapy, tumour size or the formation of tumour prostanoids.


The responses of SiHa tumours, which have essentially no transient hypoxia, were compared to the responses of WiDr tumours, which contain chronically and transiently hypoxic cells. We found that relatively modest increases in net tumour perfusion increased tumour cell radiosensitivity in WiDr tumours to a greater extent than in SiHa tumours. Moreover, redistribution of blood flow within WiDr tumours was observed on a micro-regional level that was largely independent of changes in net tumour perfusion. Through fluorescence-activated cell sorting coupled with an in vivo–in vitro cloning assay, increases in the radiosensitivity of WiDr tumour cells at intermediate levels of oxygenation were observed, consistent with the expectation that a redistribution of tumour blood flow had increased oxygen delivery to transiently hypoxic tumour cells. Our data therefore suggest that drug-induced changes in tumour micro-perfusion can alter the radiosensitivity of transiently hypoxic tumour cells, and that increasing the radiosensitivity of tumour cells at intermediate levels of oxygenation is therapeutically relevant. © 2001 Cancer Research Campaign


In vitro studies have demonstrated the relationship between the Topo IIα expression level and chemosensitivity of target cancer cells. To verify this effect in vivo, we selected 125 patients presenting with T2>3 cm and T3 N0–1 M0 breast tumours who were treated by six cycles of primary chemotherapy, including epirubicin before any surgery. Therapy response was assessed by clinical and X-ray mammogram measurements of tumour shrinkage. The pretherapeutic core biopsies were immunostained with a monoclonal antibody (Ki-S7) against Topo IIα. Ki-S7 positivity ranged from 0 to 50% . A high percentage of Ki-S7-positive cells (>15%) was associated with tumour regression under chemotherapy . Ki-S7 further emerged as an independent predictor of tumour regression , together with tumour size of less than 40 mm  and negative oestrogen receptor (ER) status , in a multivariate analysis including tumour size, SBR grade, ER and PR status, Ki-67, p53 and Her-2/neu. Our clinical results confirm in vitro data on the relationship between Topo IIα expression and tumour chemosensitivity and thus may have important practical implications.The  MCF7 cell pellets with a high percentage of cells in G2 phase were fixed in Holland's fluid and embedded in paraffin to serve as positive controls in each series assayed immunohistochemically for Topo IIα expression. Negative controls consisted of normal nonhyperplastic epithelial cells present in terminal ductal lobular units in the breast core biopsies.Ki-S7 was used undiluted as a lyophilised cell culture supernatant reconstituted with 5 ml HAll slides were read by one of the authors (GMG) who was blinded to the clinical results. Only unequivocal nuclear staining of invasive tumour cells was scored as positive  grade. The relation between Ki-S7 and the patient's age as well as tumour size was analysed by Student's t-test. A Kendall τ rank correlation test was performed to study the relationship between Ki-S7 and Ki-67, considering them as continuous variables.The χ2 test. Reciprocal influence among the different predictive factors was determined by multivariate analysis using a logistic regression test for multiple proportional hazards. The variable to predict was tumour regression ⩾50%, including complete tumour regression. To evaluate the predictive impact of Ki-S7 expression in this series, a first logistic regression model was performed (model 1), including all previously analysed factors without Ki-S7 , the survival duration was calculated from the randomisation date to death, or the date they were last known alive. All causes of death were considered as events. For metastasis-free interval (MFI) and disease-free interval (DFI), time to failure was computed from the randomisation date until metastasis or relapse, or the date they were last known to be disease-free, respectively. The median follow-up of patients in this series was 142 months, range 101–172 months.Univariate and multivariate analyses (logistic regression model) were performed using version 10.0 of SPSS software.Invasive carcinoma cells were present in 125 out of 128 biopsies; three core biopsy paraffin blocks contained no residual tumour material after tissue sectioning for histological diagnosis and analysis of the other immunohistochemical factors in the series, and the corresponding cases were excluded from the analysis. Patients' age in this series ranged from 31 to 70 years . The mean and median tumour sizes were 43.2 and 40 mm, respectively (range: 20–80 mm). The majority of tumours in this series were invasive ductal carcinomas of no special type , followed by invasive lobular carcinomas  and one mucous carcinoma (0.6%).α expression.Ki-S7 positivity ranged from 0 to 50% with a median value of 15% positive tumour cells. 15% was consequently chosen as the threshold value between low (⩽ 15%) and high (>15%) Topo IIP=1 × 10−6. and 0.03, respectively) and negatively with the immunoexpression of ER and PR . No significant association was found between Ki-S7 and Her-2/neu or tumour size  and associated (P=4 × 10−7), considering them as continuous or dichotomous variables, respectively.Ki-S7 and Ki-67 were strongly positively correlated (P=0.004) by univariate analysis (neu (P=0.092).High expression of Ki-S7 (>15%) was associated with tumour regression ⩾50% including complete tumour regression after six courses of chemotherapy .α and tumour chemosensitivity, were mainly based on in vitro experiments analysing the efficacy of other Topo IIα inhibitors in various tumour cell lines, in particular etoposide  and chromogenic in situ hybridisation (CISH) , the effect of Topo IIα inhibitors may be different in primary tumours and their corresponding asynchronous metastases, especially if an adjuvant protocol comprising a Topo IIα inhibitor had previously been applied.α .In line with the proliferation-specific expression of Topo IIα , we obseTOP2α gene . In a previous study on 863 primary operable invasive ductal carcinomas with long-term follow-up (α inhibitors, high Topo IIα expression consequently emerged as an important independent predictor of adverse outcome next to nodal metastasis and before other classical or immunohistochemical factors, including Ki-67. However, no such correlation between Topo IIα and survival was found in the present series. This could be attributable to a balanced effect of the Topo IIα expression portending increased chemosensitivity to Topo IIα inhibitors on the one hand and a higher malignant potential on the other (Contrary to previous results from our group ( was low . This fiin vivo assessment of tumour sensitivity to different chemotherapeutic drugs. Clinical decisions concerning the choice of cytotoxic therapy regimens might be improved according to the initial tumour response (α, we have shown that tumours expressing high levels of Topo IIα responded better to an anthracycline-based cytotoxic protocol than tumours with low Topo IIα expression. Therefore, we conclude that Topo IIα assessment may be of value for the clinical management of breast cancer in the setting of primary chemotherapy, provided that our results are corroborated by further studies.Primary chemotherapy in breast cancer enhances the rate of breast conservation and enables the response . With th


We have previously reported that amifostine potentiates the anti-tumour activity of carboplatin in mice. The present study was carried out in well-established human ovarian cancer xenografts OVCAR-3, A2780 and FMa grown subcutaneously in the nude mouse. It was found that a single dose of amifostine resulted in a higher increase in the anti-tumour activity of carboplatin than three doses of amifostine. A single dose of amifostine increased the AUC (area under the curve) values of total platinum in plasma ultrafiltrate (30.1 vs 18.2 microM x h), liver (307.7 vs 236.4 nmol g(-1) x h), kidney (500.8 vs 368.3 nmol g(-1) x h) and OVCAR-3 tumour tissue (184.0 vs 146.8 nmol g(-1) x h). Despite this increase in total platinum, a decrease in platinum (Pt)-DNA adduct levels was observed in liver, kidney and bone marrow, which was significant in liver. In tumour tissue an insignificant increase in Pt-DNA adduct levels, specifically the Pt-GG adduct, was observed after treatment with a single dose of amifostine, which may explain the increase in anti-tumour activity. The increase in the AUC of total platinum was probably caused by a reduction in body temperature, which was most severe after three doses of amifostine. The extreme hypothermia may be the reason that three doses of amifostine resulted in less potentiation of the efficacy of carboplatin.


Prior splenectomy increased the resistance of BALB/c mice to a syngeneic methylcholanthrene-induced ascitic tumour inoculated i.p. The survival rate of splenectomized mice was 81-6% while those of normal and sham-operated controls were 11-5% and 20% respectively. The effect of splenectomy, however, was seen only within the dose range of 10(3) to 10(4) tumour cells. This effect of splenectomy was abolished by the transfer to mice of serum from tumour-bearing mice, and of spleen cells from normal donors, immediately after the inoculation of tumour cells. Cell-free ascitis fluid did not abolish the effect of splenectomy. The findings suggest that there is a subpopulation of spleen cells which produces a tumour growth enhancing factor which is found in the serum of tumour-bearing mice.


In this period 15 351 dogs were admitted to the Clinic of Dogs and Cats and 2 145 dogs were examined in the cardiological outpatient clinic for dogs. This tumour is located in a typical place - at the base of the heart. Most frequently the tumour manifested in older boxers. Only in one case such a tumour was diagnosed in another breed of dogs. The tumours ranged in size between 3 and 16 cm in diameter. The principal sign accompanying tumours of cardiac base involved dyspnoea but in 3 cases the tumours yielded no clinical signs. All the diagnoses were additionally verified using immunohistochemical examination. We used antibodies to chromogranin A (clone DAK-A3 1:100), synaptophysin (clone SY38 1:20) and neuron-specific enolase (clone BBS/NC/VI-H14 1:150). An immunohistochemical examination is vital for the diagnosis since it allows to differentiate histologically distinct types of neoplasia which may locate in the same site and may manifest a similar histological pattern.In the period of 3 years, 9 tumours of  Chemodectomas s. paragangliomas represent tumours derived from chemoreceptor cells originating most frequently from aortic body or carotid glomus [Chemodectoma may manifest traits of a malignant tumour or of a benign tumour [chemodectoma metastases are infrequently encountered [chemodectoma tumour cells, even if DJ Meutena is his book Tumours claims that in animals chemodectomas do not produce catecholamins [chemodectoma [d glomus . The sitd glomus . Chemoden tumour ,3. It shountered . Tumour ountered ,6. The aholamins . Catechoodectoma . Diagnosodectoma . Their aodectoma ,10-13.chemodectoma type cardiac base tumours.The present study aimed at analysis of manifestation and characteristics of 2O2. Detection of chromogranin A, synaptophysin and neuron-specific enolase antigen expression was preceded by 15 min exposure of the sections in a microwave oven to a boiling Antigen Retrieval Solution  at 250 W. For demonstration of chromogranin A, synaptophysin and neuron-specific enolase antigen expression in the paraffin sections, antibodies were used in the following concentrations: clone DAK-A3 (1:100) ; clone SY38 (1:20) ; clone BBS/NC/VI-H14 (1:150) . The antibodies were diluted in the Antibody Diluent, Background Reducing solution . The sections were incubated with an antibody for 1 h at room temperature. Subsequently, incubations were performed with biotinylated antibodies  and with streptavidin-biotinylated peroxidase complex  . DAB  was used as a chromogen . All the sections were counterstained with Meyer's hematoxylin. In every case controls were included, in which specific antibody was substituted by the Primary Negative Control .The studies were performed on 9 dogs, patients of the Department of Internal Diseases with Horses, Dogs and Cats Clinic, and the Department and Clinic of Surgery, Veterinary Medicine Faculty, University of Environmental and Life Sciences. In 6 dogs the reason for the consultation of veterinary doctor was a pronounced dyspnoea. In 2 dogs the tumour of the cardiac base was diagnosed during cardiological examination, performed before surgical procedure. In one dog the tumour was detected during radiological examination of the chest, performed due to suspected fracture of the ribs following a traffic accident. In all the dogs morphology and biochemistry of venous blood was carried out, gasometric tests on arterial blood, echocardiography of the heart, chest X-ray and ECG examination were performed. All the dogs were subjected to euthanasia in the period ranging from 3 days to 18 months following the diagnosis of the cardiac base tumour. During autopsy, samples of the tumour, myocardium of the right and the left atrium, the right and the left ventricle, intraventricular septum, lungs, liver and kidneys were taken and fixed in a buffered 7% solution of formalin. Staining with hematoxylin and eosin was performed and, then, immunohistochemical studies were conducted with the use of antibodies to chromogranin A, synaptophysin and neuron-specific enolase (NSE). The sections were mounted on Superfrost slides , dewaxed with xylene and gradually rehydrated. Activity of endogenous peroxidase was inhibited by 5 min exposure to 3% HDepending on the value of proliferation index, cell morphology, possible infiltration into interior of vascular wall or myocardium as well as presence or absence of metastases the tumours could be categorized to three grades of malignancy: I (benign) - low proliferation index (up to 2 mitotic figures per 1000 cells), relatively uniform cells, no infiltration of the neighbourhood and no metastases, II  - low to average proliferation index (2 to 10 mitotic figures), less uniform cells, presence of tumour regions with infiltrative growth, possible presence of metastases, III  - high proliferative index (more than 10 mitotic figures), evident cellular pleomorphism, infiltrative growth, presence of metastases [+, Na+, Ca2+) detected no abnormalities although deviations were noted by gasometry  or malignant (4 cases) form  Table , in 6 cary Table , they mary Table . In a sir benign  cases orIn order to verify the results of the standard histopathological examination with hematoxylin-eosin staining, immunohistochemistry was applied with use of a panel of antibodies, specific for tumour cells of neuronal or neuroendocrine origin -14. All chemodectoma was confirmed in 9 dogs, accounting for 5.4% of the examined population and for 12.7% of the examined dogs with cardiac disease. A single dog with the diagnosed tumour of the cardiac base of chemodectoma has represented another breed (a dachshund). In the years 2006-2008, 1472 dogs of the dachshund breed were examined in our clinic, in that group 617 dachschunds manifested cardiac disease . In this time, among other animals, 896 German Sheppard Dogs, 267 Dobermans, 594 Yorkshire Terriers were consulted in the clinic. They are the most popular breeds in Poland. Manifestation of tumours of this type in boxers may be linked to the type of structure in the upper respiratory tract . The primary features of BAS include stenotic nares, an elongated soft palate, and distortion of the pharyngeal soft tissues due to restral shortening of the skull. These abnormalities are seen in brachycephalic breeds and they increase resistance to airflow in the upper respiratory tract. The increased negative pressure required during inspiration may result in secondary changes including laryngeal collapse. These abnormalities cause the chronic hypoxia and are suspected to provide a cause of hyperplasia and, finally, neoplasia of chemoreceptor cells [chemodectoma. It is related to chronic hypoxia. In cattle living at high altitude Ariss-Stella and Valcarcel observed carotid bodies with extreme chief cell hyperplasia and the chemodectoma was present in 40% of the animals [chemodectoma. In the dog of the dachshund breed a probable cause of poor gas exchange involved nasal polyps. It should be added that in other brachycephalic breeds, such as, e.g., Pekinese the tumour does not develop more frequently than in general population of dogs but an increased incidence of the tumour was described in Boston terriers and, thus, a family predisposition to development of chemodectoma is possible [chemodectoma contain secretory granules with catecholamines which may induce ventricular arrhythmias. In the studied dogs with chemodectoma numerous premature ventricular beats were detected. One dog manifested attacks of ventricular tachycardia, which disappeared following intravenous administration of a beta-blocker but did not vanish following administration of lidocaine, which is the drug of choice. This might be interpreted as consistent with the potential for production of catecholamines by chemodectoma tumours. The described location of the tumour might predispose to supraventricular arrhythmias which, however, were not recorded in the examined dogs. Blumcke et al examined reaction of carotid bodies in rats to acute and severe oxygen deficiency. They detected the almost total discharge of catecholamines from type I cells after 20 minutes of extreme hypoxia, which was confirmed by fluorescence microscopy. Mitchell postulated that catecholamines in the type I cells may function as transmitters in an efferent neuronal pathway. Chemodectoma represents a tumour which usually does not metastasize. Nevertheless, some cases of metastases were described - to lungs, liver, myocardium and even to brain and bones [Tumours of the cardiac base usually develop in older animals ,15. In tOut of all the applied markers, used to detect neoplastic cells of neuronal or neuroendocrine origin, the highest significance was linked to chromogranin A. The protein, as a protein prohormone, represents a marker of neuroendocrine differentiation and the directed against it antibodies are used to identify cells and tumours of neuroendocrine origin. Expression of NSE and that of synaptophysine manifested a similar level in the cells of both benign and malignant tumours. On the other hand, in the case of chromogranin A we, similarly to Aresu et al. , have foOur observations have shown that all the applied antibodies are useful in differential diagnosis of neoplastic processes located at the cardiac base but only chromogranin A may help in evaluation of tumour malignancy.chemodectoma develops in dogs of the boxer breed significantly more frequently than in the general population of dogs. As demonstrated by our observations, the sensitive examination, which allows to detect them, involves the radiological examination of the chest. The examination of the fluid drained from the pleural or pericardial cavity is useless in the diagnosis of chemodectoma since tumour cells are not des quamated to the fluid. Gasometric blood tests may be helpful in selecting boxers with the risk of chemodectoma development. The diagnosis requires confirmation by the histopathological examination.Summing up, The authors declare that they have no competing interests.ANN carried out ECG and echocardiographic examinations, blood collection and interpreted the tests, drafted the manuscript. UP carried out echocardiographic examinations. MN performed the histopathological examinations and drafted the manuscript. WA carried out X-ray examinations. JN interpreted blood analysis and drafted the manuscript. All authors read and approved the final manuscript.


In confirmation of other studies it has been shown that antibody directed against the tumour specific transplantation-type antigens (TSTAs) cannot be detected in rats with a tumour growing intramuscularly but appears within a few days after excision of the tumour. Circulating antibody is found in the serum of rats with lymph node metastasis following excision of the primary. Absorption by the intramuscular tumour of circulating antibody does not account for the absence of antibody since the antibody levels in thoracic duct lymph in rats with tumours in the leg are also very low and rise rapidly after tumour excision. Antibody is released by the draining nodes directly into the lymph and must pass through the thoracic duct before entering the blood. Under these conditions low levels of antibody activity in lymph cannot be ascribed to absorption by the tumour.It is postulated that TSTA is released from the tumour into the lymph. Following injection of tumour cells into immunized rats the level of antibody falls and this is attributed to release of TSTA from the injected cells. The possible role of antigen release from tumours in determining the host reaction to the tumour is discussed.


A substance capable of inducing tumour cell aggregation, which is supposed to be a glycoprotein showing noncytotoxicity, was separated from rat ascites hepatoma cells and partially purified by chromatography. Adhesiveness of rat ascites hepatoma cells induced by this substance was characterized by gradual development of known binding structures during a period of 24 h after contact with the substance; simple apposition and intermediate junctions developed in the early stage, and desmosomes and focal tight junctions in the later stage. It was assumed that the substance might be involved in the development of such binding structures as a triggering mechanism of tumour cell adhesiveness.


A rapidly growing human squamous cell carcinoma (FaDudd) was used for the study. For experiments, NMRI (nu/nu) mice with subcutaneously growing tumours were randomly allocated to no treatment, Mitomycin C, fractionated irradiation (ambient: 11x4.5 Gy in 15 days), or fractionated irradiation combined with Mitomycin C. Graded top up doses (clamped blood flow: 0–57 Gy) were given at day 16, 23, 30 or 37. End point of the study was the time to local tumour progression. Data were examined by multiple regression analysis (Cox). Mitomycin C alone resulted in a median time to local tumour progression of 23 (95% confidence limits: 17–43) days, fractionated irradiation in 31 (25–35) days and combined Mitomycin C plus fractionated irradiation in 65 (58–73) days (P=0.02). Mitomycin C decreased the relative risk of local recurrence by 94% (P<<0.001) equivalent to 31.7 Gy top up dose. Repopulation accounted for 1.33 (0.95–1.72) Gy per day top up dose after fractionated irradiation alone and for 0.68 (0.13–1.22) Gy per day after fractionated irradiation+Mitomycin C (P=0.018). Mitomycin C significantly reduces the risk of local recurrence and inhibits tumour cell repopulation in combination with fractionated irradiation in vivo in the tested tumour model.The potential of Mitomycin C in combination with fractionated irradiation to inhibit tumour cell repopulation of a fast growing squamous cell carcinoma after fractionated radiotherapy was investigated British Journal of Cancer (2002) 86, 470–476. DOI: 10.1038/sj/bjc/6600081www.bjcancer.comThe Cancer Research Campaign© 2002  Repopulation of surviving clonogenic tumour cells during fractionated radiotherapy has been identified as an important factor associated with clinical failure in squamous cell carcinoma of the head and neck . AccordiSquamous cell carcinomas of the head and neck have been shown to be a chemosensitive disease. Cisplatin and taxane based regimens in combination with 5-fluorouracil render overall response rates of 80–90% in locally advanced disease , the killing of hypoxic tumour cells . The impdd was used for the study. The cell line was kindly provided by M Baumann . FaDudd is a well characterized subline derived from the ATCC cell line FaDu. The DNA content is aneuploid and the p53 tumour suppressor gene is mutated. FaDudd exhibits rapid growth in vitro and in vivo on nude or SCID mice  -nude mice were bred in a specific pathogen free animal colony at the University of Essen. At an age of 4–6 weeks, animals were brought to Tuebingen University and housed in an individually ventilated cage rack system . They were fed sterile high-calorie laboratory food and drank water supplemented by chlorotetracycline and potassium sorbate acidified to a pH of 3.0 with hydrochloric acid 3 the animals were randomly allocated to the following treatment arms  (n=11); (b) fractionated radiotherapy (11×4.5 Gy in 15 days) followed by graded top up doses (0–57 Gy) at day 16, 23, 30 or 37  (n=117); (c) fractionated radiotherapy (11×4.5 Gy in 15 days) in combination with MMC on day 1 and 8  followed by graded top up doses (0–37 Gy) at day 16, 23, 30 or 37  (n=139); (d) no treatment  (n=25).For the experiments a source tumour was excised and tumour chunks of about 2 mm diameter were implanted subcutaneously into the right hind limb of 6–10-week old animals. Wound closure was not necessary. Approximately 2–3 weeks after transplantation visible tumour growth occurred. The tumour size was scored three times a week. When the tumours reached a volume of approximately 120 mm3 (standard deviation 39.1 mm3). According to the results of previous experiments (3 at the time of the last top up dose (day 37). At larger sizes, tumours tend to spread to the trunk of the mice and cannot be sufficiently irradiated to achieve long term local tumour control; (b) Long term local tumour control must not be attained by fractionated radiotherapy alone. Top up doses with graded doses were administered at different intervals after fractionated radiotherapy in order to estimate isoeffective dose levels and calculate the dose needed to counteract repopulation during the interval between fractionated radiotherapy and top up irradiation (with and without MMC).Experiments were performed in two runs. The first run consisted of 144 and the second run of 148 randomized animals. The median tumour volume at the start of treatment was 126 mm−1 dose rate) with an experimental device as described earlier  intraperitoneally approximatley 1 h before radiation therapy on days 1 and 8 of the treatment. The injected volume was 0.1 ml per 10 g body weight. Animals randomized to radiotherapy alone got no injections.Animals randomized to chemotherapy received 3 mg kg2)/2, where a and b are the long axis and the short axis, respectively. Scoring of tumour sizes took place three times per week before start of treatment and twice per week after the start of the treatment. The investigators were not blinded as to the treatment that the animals had received, when they made their measurements. Follow up was terminated at day 280 or in case of intercurrent death or if recurrent tumours after top up irradiation had grown to eight-times the initial tumour volume at the start of treatment. Body weight was recorded once a week. A modified growth delay end point, time to local tumour progression (TTP), was end point of the study. For animals randomized to receive MMC only or fractionated radiotherapy alone without top up doses, the TTP was defined as interval between the day of start of treatment to the day when the regrowing tumours reached twice the initial tumour volume. For animals randomized for a top up irradiation, the TTP was defined as interval between the day of start of treatment to the day after top up irradiation when the regrowing tumours reached twice the initial tumour volume.Tumour size was measured with calipers in two perpendicular diameters. The tumour volume (V) was calculated as V=(a×bn=256). Tested parameters were top up dose (0–57 Gy), MMC (yes or no), time of top up radiotherapy (0–3 weeks after fractionated radiotherapy), and MMC×(time of top up radiotherapy). The relative risk ratios derived from the Cox model were the basis for calculation of the radiotherapy top up dose equivalents for the effect of MMC and time effect with and without MMC.The median TTP was calculated according to the method of Acute toxicity as measured as maximal weight loss of the animals during the fractionated radiotherapy (±MMC) was only moderate. Mice receiving radiotherapy alone and radiotherapy in combination with MMC exhibited a maximal median weight loss of 10% (s.d.±6%) and 6% (s.d.±5%), respectively (not significant). The nadir of weight loss occurred at day 9 in the radiotherapy alone arm of the study and at day 8 in the radiotherapy+MMC arm of the study.During longer follow-up after fractionated radiotherapy intercurrent deaths occurred as a result of the limited life span of nude mice. However, the frequency and time of intercurrent deaths did not significantly differ between animals treated with and without MMC  compared to 4.5 (CL: 3.2–5.6) days (n=25) in untreated control animals (P<0.001). After fractionated radiotherapy alone (without top up dose) a median TTP of 31.0 (CL: 25.0–34.9) days was observed compared to 64.9 (CL: 57.9–72.8) days after fractionated radiotherapy in combination with MMC (P=0.02). The difference in TTP after MMC alone (23.0 days) and fractionated radiotherapy alone (31.0 days) was not significant.MMC alone exhibited moderate tumour activity resulting in a median TTP of 23.0 (95% confidence limits (CL) 17.0–42.8) days , although the median top up doses were 15 Gy lower in the MMC arm (30 Gy vs 15 Gy)  in the radiotherapy alone arm  and in 44 out of 139 animals (31.7%) in the radiotherapy+MMC arm  of the study .The results of the multivariate analysis of TTP is shown in Table 1P<0.001, Mann–Whitney U-test). A non-significant trend towards longer TDT of recurrent tumours were observed with increasing top up doses especially after treatment with MMC : 13.7 days, 95% CL: 11.9–15.6 days) compared to untreated control tumours . TDT of recurrent tumours that had received MMC during fractionated radiotherapy were significantly longer (17.4 days (95% CL: 14.3–20.6 days)) than TDT of recurrent tumours that had not received MMC (11.0 days (95% CL: 9.6–12.4 days)) . Repopulation in the interval between fractionated radiotherapy and top up irradiation accounted for 1.33 Gy per day top up dose after fractionated radiotherapy alone and for 0.68 Gy per day after fractionated radiotherapy in combination with MMC  and for 23 Gy top up dose or for 50% of the MMC effect at the last top up irradiation (day 37). Translated to a 6–7 weeks clinical radiation series in head and neck cancer, this would mean that inhibition of repopulation by MMC  might be of major importance for the observed beneficial effects of simultaneous chemo-radiotherapy. This would only be true, if repopulation is inhibited more in the tumour than in normal tissues. A significant inhibition of repopulation in normal tissues by MMC should be clinically detectable by an enhanced radiation mucositis and dermatitis. However, in clinical trials on head and neck cancer using MMC, only little or no evidence of enhanced mucosal or dermal side effects was found, whereas locoregional tumour control was considerably increased  was observed for the chemo-radiation arm of the study. The extent of the observed survival benefit is not smaller than in other chemo-radiation trials using identical overall treatment times or accelerated treatments in both study arms  so that we do not know whether our observations will be typical for other tumour cell lines and cytostatic drugs. Large differences in the sensitivity of human tumours towards MMC have been observed. The tumour response is influenced by the reductive enzyme profile of the tumour (But despite these critical appointments we demonstrated, as a proof of principle that chemotherapy, especially MMC, is able to inhibit tumour cell repopulation significantly. Inhibition of repopulation appears to be a potentially important mechanism for the beneficial effects of simultaneous chemo-radiation. An accelerated radiotherapy might not be necessary if an effective chemotherapy is used concurrently. This issue deserves further experimental and clinical investigations.


Photodynamic therapy (PDT) may cause tumour cell destruction by direct toxicity or by inducing cellular hypoxia as a result of microcirculatory shutdown. Aminolaevulinic acid (ALA) causes cellular accumulation of protoporphyrin IX (PPIX) in cells exposed to it in excess. PPIX can be used as a photosensitizer for PDT. Microcirculatory shutdown may be induced by toxicity to the endothelial and vascular smooth muscle (VSM) cells or by release of vasoactive substances. We have studied whether PPIX is produced by endothelial, VSM and tumour cells on exposure to ALA and whether these cell lines are directly damaged by PDT in vitro. Tumour endothelial cells are angiogenic and we have, therefore, investigated the effect of cellular proliferation rates on PPIX generation. Tumour cells generate more PPIX intracellularly than the non-neoplastic cell lines studied and are correspondingly more sensitive to PDT-induced cytotoxicity. Endothelial cells are sensitive to PDT-induced cytotoxicity and accumulate between 1.5 and four times more PPIX when proliferating (as during tumour-induced angiogenesis) than when quiescent. We conclude that PPIX-mediated PDT may exert some of its effects on the microcirculation of treated tissues by direct toxicity to endothelial and VSM cells, and that this toxicity may be enhanced in the tumour microenvironment.


Polyethylene glycol (PEG) modification of a chimeric Fab' fragment (F9) of A5B7 , using an improved coupling method, increases its specificity for subcutaneous LS174T tumours. PEGylation increased the area under the concentration-time curve (AUC0-144) in all tissues but there were significant differences  between the proportional increases in AUC0-144, with the tumour showing the greatest increase. The increase in AUCtumour from F9 to PEG-F9 was similar to the reported increase from Fab' to F(ab')2 while the increase in AUCblood by PEGylation of F9 was only 21% of the reported increase from Fab' to whole IgG. A two sample t-test showed no significant differences between maximal tumour/tissue ratios for PEG-F9 and F9 while the tumour/tissue ratios for PEG-F9 remained high over a longer period, with tumour levels at least double those for F9. PEG-F9 emerges as a new generation antibody with potential advantages for both radioimmunotherapy and tumour imaging. Since there was a reduction in antigen binding, optimisation of PEGylation might further improve tumour specificity. The latter resulted from complex effects on both the entry into and exit rates from tumour and normal tissues in a tissue-specific fashion.


Double vaccinations enhanced the anti-tumour effect in all groups except the intravenous (i.v.) group, which failed to achieve complete rejection. The anti-tumour efficacy of each immunization route was correlated with the OVA-specific cytotoxic T lymphocyte (CTL) activity evaluated on day 7 post-vaccination. Furthermore, the accumulation of DC2.4 cells in the regional lymph nodes was detected only in the i.d.-and s.c.-injected groups. These results demonstrate that the administration route of antigen-loaded DCs affects the migration of DCs to lymphoid tissues and the magnitude of antigen-specific CTL response. Furthermore, the immunization route affects vaccine efficiency. © 2001 Cancer Research Campaign http://www.bjcancer.com Dendritic cells (DCs) loaded with tumour antigens have been successfully used to induce protective tumour immunity in murine models and human trials. However, it is still unclear which DC administration route elicits a superior therapeutic effect. Herein, we investigated the vaccine efficiency of DC2.4 cells, a murine dendritic cell line, pulsed with ovalbumin (OVA) in the murine E.G7-OVA tumour model after immunization via various routes. After a single vaccination using 1 × 10


Samples of Walker 256 carcinoma grown in muscles of Sprague-Dawley rats were studied at low temperatures before and after lyophilization. The effects of lyophilization on the ESR spectra were different for tumours and normal muscle. Prior to lyophilization of a tumour sample, there was a decrease in free radicals, while after the lyophilization, there was an "increase". The "increase" was due to the lyophilized tumour having a narrower line, producing a greater peak-peak height measurement than in muscle, without an increase in the total number of spins. Exposure of lyophilized samples to air produced an increase in the intensity of the spectra and a change in line shape; also these effects differed for tumour and muscle. Mn++ levels were lower in tumour than in muscle, a difference eliminated by lyophilization. Poor growth conditions in tumours increased the occurrence of ESR spectra due to NO complexes with both heme and non-heme iron proteins. These results may help to resolve the principal controversies about experimental findings in ESR of tumours. At least part of the signals seen after lyophilization do not reflect free radicals in vivo. The signals after lyophilization reflect biochemical differences between tumour and muscle; spectroscopic data indicate that it is feasible to determine the molecular basis of these differences.


One reason why photodynamic therapy has not been considered suitable for treatment of metastatic tumours in the liver, is the poor selectivity of conventional photosensitizers for tumour compared to normal liver. This report details an alternative approach to increasing this selectivity by the use of antibody-targeted photosensitizers (or photoimmunoconjugates) to target intrahepatic tumours caused by human colorectal cancer cells in the nude mouse, and explores the role of molecular charge on the tumour-targeting efficiency of macromolecules. The murine monoclonal antibody 17.1A (which recognizes an antigen expressed on HT 29 cells) was used to prepare site-specific photoimmunoconjugates with the photosensitizer chlorine6. The conjugates had either a predominant cationic or anionic charge and were injected i.v. into tumour-bearing mice. Biodistribution 3 or 24 h later was measured by extraction of tissue samples and quantitation of chlorine6 content by fluorescence spectroscopy. The photoimmunoconjugates were compared to the polylysine conjugates in an attempt to define the effect of molecular charge as well as antibody targeting. The anionic 17.1A conjugate delivered more than twice as much photosensitizer to the tumour at 3 h than other species (5 times more than the cationic 17.1A conjugate) and had a tumour:normal liver ratio of 2.5. Tumour-to-liver ratios were greater than one for most compounds at 3 h but declined at 24 h. Tumour-to-skin ratios were high (> 38) for all conjugates but not for free chlorine6. Cationic species had a high uptake in the lungs compared to anionic species. The photoimmunoconjugates show an advantage over literature reports of other photosensitizers, which can result in tumour:normal liver ratios of less than 1. © 2000 Cancer Research Campaign