To enhance the success rate of nanocarrier-mediated chemotherapy combined with an anti-angiogenic agent, it is crucial to identify parameters for tumour vasculature that can predict a response to the treatment of the anti-angiogenic agent.β type I receptor (TβR-I) inhibitor, A-83-01, to combined therapy, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was carried out in mice bearing colon 26 cells using gadolinium (Gd)-DTPA and for its liposomal formulation to evaluate changes in tumour microvasculature following A-83-01. Tumour vascular parameters from DCE-MRI were compared with histological assessment and apparent diffusion coefficient of water in tumour generated by diffusion-weighted MRI.To apply transforming growth factor (TGF)-60), volume transfer constant (Ktrans) and fractional plasma volume (vp) significantly within 24 h, that was positively related to α-smooth muscle actin-positive pericyte coverage and tumour cell proliferation, and was correlated inversely with the apparent diffusion coefficient. The vascular function of the tumour improved by A-83-01 treatment was well assessed on post-liposomal Gd-DTPA-enhanced MR images, which predicted delivery of a liposomal drug to the tumour.Contrary to evaluations reported for anti-angiogenic agents, A-83-01 treatment increased the initial area under the Gd concentration–time curve (IAUGCKtrans and vp quantitation, provide important additional information about tumour vasculature by A-83-01 treatment.These findings suggest that DCE-MRI and, in particular,  The success of chemotherapeutic agents with solid tumours is critically dependant on the access that these agents have to the tumours via the so-called ‘leaky vasculature’. In particular, tumour vasculature is crucial for the delivery of drugs encapsulated in nanocarriers . Anti-anβ in cancer biology are complex; TGF-β can suppress or promote tumour growth depending on the type of cancer. Small molecule TGF-β type I receptor (TβR-I) inhibitor has a wide variety of effects -5 (IC50=12 nM) (50=59 nM) -vp) and the volume transfer constant (Ktrans) from the enhancement of tumour signal intensity by gadolinium (Gd) contrast agent  is one of the evaluation methods of anti-angiogenic agents, such as anti-VEGF antibody and tyrosine kinase inhibitor, clinically  were inoculated subcutaneously into the right back at the side of the heart in CDF1 female mice . When the tumour size reached approximately 100 mm3, A-83-01    dissolveat ‘0 h’ . In thisinductively coupled plasma with an SPS7800 apparatus . T1 relaxation times of Gd-L and Gd-DTPA were measured over the concentration range of 0–0.25 mM Gd at 9.4 T1 at room temperature. Relaxivity (R1) was then determined from the slope of the linear regression fits of 1/T1vs the Gd concentration: 1/T1=R1 × [Gd]+1/T10, where T10 represents T1 of 0 mM Gd solution. R1 of Gd-L was 4.48 mM−1s−1, which was similar to that of Gd-DTPA (4.39 mM−1s−1).For the preparation of liposomal Gd-DTPA (Gd-L), mixture of egg phosphatidylcholine , cholesterol , and polyethyleneglycol 2000-distearoyl phosphatidylethanolamine  in a molar ratio of 5 : 2 : 0.35 was dissolved in ethanol at 60°C, hydrated with Gd-DTPA , stirred, and evaporated under a vacuum to remove ethanol. This mixture was exposed to ultrasound until the particle diameter was about 120 nm, followed by exhausted dialysis against phosphate buffered saline (pH 7.4) solution. The particle size of the liposomes was determined at 25°C using an ELS-Z2 instrument . The Gd concentration was determined using Apparent diffusion coefficient was estimated and mapped from diffusion-weighted imaging using the following parameters: repetition time (TR)=2000 ms, echo time (TE)=45 ms, slice thickness 3 mm, 64 × 64 data matrix, axial orientation, and field-of-view of 3 × 3 cm2. Three slices through the centre of the tumour were acquired. Diffusion gradients equivalent to b-values of 0, 200, 400, and 800 s mm−2 were employed using gradient pulse widths of δ=7 ms and Δ=20 ms.2-weighted spin–echo axial images were acquired to detect the tumour position. Pre-contrast tumour T1 was determined using an inversion recovery-prepared spoiled gradient-recalled echo (SPGR) sequence. The inversion-recovery was carried out using a 180° hard RF pulse followed by a gradient crusher pulse. Inversion times were 0.2, 0.4, 0.8, 1.4, 2, and 3 s. The other MRI parameters were: TE=3 ms, field-of-view=3 × 3 cm2, slice thickness=4 mm, and matrix size=64 × 64. Both RF and gradient spoilers were applied. In DCE-MRI acquisition, it was applied repeatedly to acquire the axial slice SPGR images through the tumour and left ventricle with a second temporal resolution over 6 min: TR=7.8125 ms, TE=2.06 ms, matrix resolution=64 × 64, field-of-view=3 × 3 cm2, slice thickness=4 mm, flip angle=30°, number of slices=1, and two signal averages. Approximately 20 s of baseline DCE-MRI images were acquired. Gd-DTPA or Gd-L was administered at 20 μl g−1 (0.1 mmol Gd kg−1) as a bolus with heparinized saline  via manual injection over 2–3 s.Dynamic contrast-enhanced magnetic resonance imaging was carried out with Gd-DTPA and Gd-L before (‘pre’) and after treatment in each animal. With the use of Gd Gd-L, injected lipids containing Gd-L were retained in the tumour; therefore, different mice were used to compare pretreatment with treatment of A-83-01. Before DCE-MRI high spatial resolution, two-dimensional T2-weighted axial images, using ImageJ software , and the tumour ROI was transferred to the apparent diffusion coefficient map calculated from diffusion-weighted imagings.Tumour regions-of-interest (ROI) covering the whole tumour was segmented on the T1 map of tumour was prepared by the imaging of the inversion-recovery method to quantitate tumour Gd concentrations. The concentration of Gd at each imaging time point in each voxel was estimated using the formula 1 in blood plasma at 9.4 T was 2.2 s, as reported previously (1 in tumour was from the T1 map. 1/T1 (t) was calculated for every time point for the blood and tumour Gd concentrations . The initial area under the Gd concentration–time curve over 60 s (IAUGC60) was calculated. The tumour haemodynamic parameters Ktrans and vp were calculated using a two-compartment model (A TμM sections for hematoxylin and eosin staining and immunostaining. Antibodies against α-smooth muscle actin (SMA)  were used to identify the pericyte and anti-CD31 , endothelial cell marker and anti-Ki67  antibodies to recognise the growth state cells . Vascular areas within the tumours were measured as the index of tumour vascularity by stained with anti-CD31. Five fields of tumour sections were analysed at low magnification using a computerised image analyser . The ratio of vessel area against tumour area without necrosis was calculated.For the histological assessment of A-83-01 effects on tumour vasculature, tumour sections were observed at 24 h after repeated injection of A-83-01. Each of four tumours from A-83-01 treated and untreated mice was resected and fixed with 10% formalin. Paraffin-embedded samples were sliced into 3 t-test was used comparison between the pre- and post-treatment groups. ANOVA analysis, followed by Dunnett's test, was used for multi-group comparisons. Pearson's correlation coefficients were used for determination between a significant positive and negative relationship. Correlations between 0.4 and 0.6 were considered moderate, whereas correlations from 0.7 to 1.0 were considered strong. Significant differences were accepted when the P-value was below 0.05.Values were expressed as the mean±s.d. A two-tailed Student's 60, 60). At 24 h after repeated injection, the tumour accumulation increased a similar level to that at 3 h after the single injection (1.8-fold the IAUGC60). Next, we observed changes of the tumour vasculature repeat-treated by A-83-01 using Gd-L. The Gd concentration in DCE-MRI acquisition of untreated mice was very low  and Gd-L. At 3 h after the first treatment, vp and Ktrans did not show significantly elevated values with Gd-DTPA, therefore, it can be concluded that the repeated administration schedule changed the tumour state positively for better liposomal contrast-agent distribution. The most characteristic point of the vp and Ktrans changes was the large dispersion of vp and Ktrans values after repeated A-83-01 treatments with the use of Gd-L. The diversity of local permeability of treated tumours may lead to large dispersion of Gd-L. The mouse tumour core showed an increase in the Gd concentration, as shown in vp and Ktrans values among treated mice.From the data obtained above, lculated . With Gdμm width and 100 μm depth (P>0.05), as the change was very diverse within a tumour (P<0.05) (μm depth . The bleμm depth . Tumour a tumour . The abna tumour . It is ia tumour . The Ki6(P<0.05) . These fapparent diffusion coefficient value, single-treated groups at 3 and 24 h did not show a difference compared with the pretreatment, but the repeat-treated group at 24 h showed a significant difference  after the repeated A-83-01 treatment was higher to that with Gd-DTPA (Ktrans=0.035±0.009 min−1) (An increase in 9 min−1) . LiposomKtrans and IAUGC (Ktrans in solid tumours is concerned with the anti-tumour effect (Ktrans, IAUGC60, and vp were increased significantly 24 h after the A-83-01 treatment. This increase may be explained by different treatment protocols, different tumour models, and the different signal inhibition between anti-angiogenic agent such as kinase inhibitor and TβR-I inhibitor. Similar to A-83-01-treated colon 26 solid tumours, LY364947-treated M109 solid tumours increased IAUGC60 at 3 h and recovered fully by 24 h post-injection (Anti-angiogenic agents such as anti-VEGF antibody and tyrosine kinase inhibitor were reported that decrease both njection .Ktrans and IAUGC60 values were related to the increased number of growth state cells around the tumour vessel, and were correlated to the decreased apparent diffusion coefficient value. Because of no significant difference in tumour cell shape after treatment (apparent diffusion coefficient, therefore, reflected a decrease in extra-cellular fluid, suggesting that the recovery of delivery may be related to vessel normalisation.It was reported that in a limited situation, anti-angiogenic agents work to deliver more drugs into tumours through the induction of vascular normalisation . Untreatreatment , intra-cKtrans, and vp showed a moderate negative correlation to apparent diffusion coefficient, suggesting that these parameters may be providing similar information. As apparent diffusion coefficient is acquired in clinic widely to detect and diagnose a tumour, it could apply conveniently to examine the permeability of tumour in patients.Furthermore, similar to negative correlation between tumour interstitial fluid pressure and permeability of tumour (βR-I inhibitor-combined therapy.Although there is room for improvement, DCE-MRI using liposomal contrast agents such as Gd-L could be an important method to anticipate the extravasation of the liposomal anti-cancer drug during TKtrans, IAUGC60, and vp were positively related to tumour vasculature by the treatment of A-83-01. Thus, TβR-I inhibitor has the potential to enhance the delivery of liposomal anti-cancer drugs and contrast agents. DCE-MRI forms a capable tool to determine the administration schedule of combination therapy with TβR-I inhibitor by Ktrans and vp quantitation.In summary, we found that DCE-MRI parameters,


Here we report the treatment effect of ionizing radiation in combination with the novel orally bioavailable VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 on endothelial cell proliferation in vitro and with tumour xenografts in vivo. Combined treatment of human umbilical vein endothelial cells with increasing doses of PTK787/ZK222584 and ionizing radiation abrogated VEGF-dependent proliferation in a dose-dependent way, but inhibition of endothelial cell proliferation was not due to apoptosis induction. In vivo, a combined treatment regimen of PTK787/ZK222584 (4 × 100 mg/kg) during 4 consecutive days in combination with ionizing radiation (4 × 3 Gy) exerted a substantial tumour growth delay for radiation-resistant p53-disfunctional tumour xenografts derived from SW480 colon adenocarcinoma cells while each treatment modality alone had only a minimal effect on tumour size and neovascularization. SW480 tumours from animals that received a combined treatment regimen, displayed not only an extended tumour growth delay but also a significant decrease in the number of microvessels in the tumour xenograft. These results support the model of a cooperative antitumoural effect of angiogenesis inhibitor and irradiation and show that the orally bioavailable VEGF receptor tyrosine kinase inhibitor PTK787/ZK222584 is suitable for combination therapy with irradiation. © 2001 Cancer Research Campaign


Investigations were performed using a VEGF-secreting human head and neck tumour cell line, CAL33, with a high EGFR content, injected as orthotopic xenografts into the mouth floor of nude mice. Three days after tumour cell injection, bevacizumab , erlotinib  and irradiation  were administered alone and in combination for 10 days. As compared with the control, concomitant administration of drugs produced a marked and significant supra-additive decrease in tumour mass; the addition of irradiation almost completely abolished tumour growth. The drug association markedly reduced the number of metastatic nodes and the triple combination significantly reduced the total number of pathologically positive lymph nodes as compared with controls. The RT-induced proliferation, reflected by Ki67 labelling, was reduced to control level with the triple combination. Radiotherapy induced a strong and very significant increase in tumour angiogenesis, which was no longer observed when combined with erlotinib and bevacizumab. The efficacy of the combination of bevacizumab+erlotinib and RT may be of clinical importance in the management of head and neck cancer patients.Clinical benefit has been demonstrated in patients with head and neck tumours receiving an anti-epidermal growth factor receptor (EGFR) agent in combination with radiotherapy (RT). Recent preclinical and clinical studies suggest beneficial effects from combining anti-angiogenic drugs with RT. To investigate the effect of combining these approaches, we evaluated  Agents that target epidermal growth factor receptor (EGFR) potentially exert anti-tumour effects by inhibiting tumour cell proliferation and survival, as well as reducing the secretion of proangiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor that stimulate tumour neoangiogenesis  in nude mice. The median time to reach a tumour volume of 1000 mm3 was significantly increased for AZD2171 or gefitinib administered alone compared with the control. Greater inhibition of tumour growth was seen with the combination AZD2171+gefitinib compared with either drug alone, and the triple combination compared with either AZD2171+gefitinib or RT alone.Tumour vasculature is a key target in the treatment of solid tumours, particularly in head and neck cancer . In a reThe aim of this study was to examine the anti-tumour effects of the clinically representative anti-angiogenic monoclonal antibody bevacizumab combined with the anti-EGFR tyrosine kinase inhibitor erlotinib and RT. To this end, we adopted an orthotopic head and neck cancer xenograft model consisting in injecting CAL33, a human head and neck cancer cell line, into the mouth floor of nude mice. This model was used to enable examination of the effects of treatment on the primary tumour itself and also, for a more clinically relevant condition, on disease extension to the lymph nodes in the neck. Molecular markers were also examined according to the applied treatments. Ki67 expression was considered as a relevant and classical parameter related to tumour proliferation. The key VEGF receptor present on endothelial cells is VEGFR2. In addition, the presence of VEGFR2 has been reported on tumour cells themselves, particularly in head and neck cancer (−1) was suspended in 0.9% NaCl and 0.01% Tween 80, and bevacizumab (750 mg l−1) was diluted in 0.9% NaCl. For both drugs, the concentrations were adjusted so as to include the daily dose in 0.2 ml of drug suspension. Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin and glutamine were purchased from Whittaker . Fetal bovine serum (FBS) was obtained from Dutscher .Erlotinib was kindly provided by Roche  and bevacizumab by our institution's pharmacy. Working solutions were prepared as follows: erlotinib . This cell line exhibits high EGFR levels  at 37°C in an atmosphere containing 8% CO2. Batches of 15 × 106 cells were frozen in FBS supplemented with 5% dimethyl sulphoxide v/v in advance for injection into mice. Shortly before injection, cells were thawed and suspended in Ringer lactate.The cell line was maintained as monolayer culture in DMEM supplemented with 10% FBS v/v, 2 m6 cells suspended in 200 μl of Ringer lactate (n=10 per treatment condition). There were five animals per cage with food and water available ad libitum; animals were killed and tumours collected at day 10 after cell injection by cervical disruption . As no signs of suffering appeared during the experiment , no animal had to be killed before the end of the experiment. Animal experiments were performed in accordance with the regulations of our institution's ethics commission and guidelines of the United Kingdom Co-ordinating Committee on Cancer Research . They received, as intra-muscular orthotopic xenograft in the mouth floor as described by −1, 5 days a week, 0.2 ml p.o.), bevacizumab  and irradiation  given alone or in combination; when co-administered, erlotinib and bevacizumab were given simultaneously (given the short duration of treatment − 7 days − five doses of each drug and four doses of irradiation were delivered); the doses of erlotinib and bevacizumab were chosen according to preliminary experiments , performed on tumour of this same cell line xenografted in the flank of animals, so that each drug given alone exerted only partial effects on tumour growth.Three days after cell injection, the mice were treated each week with relevant vehicle (controls), erlotinib (50 mg kgπ/6 × length × width2) and weighed, and bilateral radical neck dissection was performed . The effects of erlotinib, bevacizumab and RT, alone or in combination, on tumour growth were evaluated by measuring, for all groups, the mean tumour volume (MTV). Combination ratios (CR), as described previously If CR>1, there were supra-additive effects and if CR<1, infra-additive ones. Strictly additive effects were observed if CR=1.The total number of pathologically positive lymph nodes, having signs of inflammation either invaded or not, having a visible diameter (greater than 1 mm), were recorded after node chain dissection after tumour removal.All analyses of tumour markers were performed on the primary tumours, which were collected 10 days after tumour cell injection.The expression of the microvessel marker VEGFR2 and of the proliferation marker Ki67 was evaluated using immunohistochemistry . The analysis of VEGFR2 involved vessel loss and reduction in vessel area, which were evaluated both at the tumour centre and tumour periphery, and also a more diffuse labelling of tumour cells; in this case, both the intensity  of labelling and the proportion of tumour cells were taken into account. The analysis of Ki67 took into account the proportion of labelled cells; labelling of each microscopic field was attributed to three labelling groups: group 1 with 15–50% of labelled cell nuclei; group 2 with 50–70% of labelled cell nuclei; and group 3 with more than 70% of labelled cell nuclei. For each treatment group, the relative proportion of each of the three labelling groups  was pooled and represented as a histogram.Forty × 10 magnification images of regions of high vascular density within the tumour were analysed to quantify both tumour angiogenesis and tumour cell diffuse labelling (VEGFR2). Whatever the studied marker (Ki67 or VEGFR2), the final score was the result of the examination of a maximum of 10, a minimum of 2 and a mean of 5 fields per tumour, which, due to the small volume of the tumour, allowed a thorough examination; 10 tumours were investigated for each treatment group.Comparison of primary tumour growth, node number, state of invasion and the effects of treatment on VEGFR-2 and Ki67 between different treatment groups were evaluated using the non-parametric ANOVA .P>0.05) (P=0.028 vs controls) and showed supra-additive effects (CR=2).Treatments given alone had no significant impact on primary tumour mass, with erlotinib and RT slightly decreasing tumour growth  . The comP<0.0004 vs controls; P<0.0001 vs RT; P=0.02 vs bevacizumab+erlotinib). The effects of this triple combination were supra-additive (CR=2.3).Finally, the triple combination bevacizumab+erlotinib+RT gave the highest tumour growth inhibition . However, the drug combination had no effect on the total number of pathologically positive lymph nodes. In contrast, the bevacizumab+erlotinib+RT triple combination produced a very significant decrease in the total number of pathologically positive lymph nodes (P<0.001 vs controls) although invaded nodes were still present among these markedly reduced pathologically positive lymph nodes.In contrast, and corroborating the observations on the primary tumour mass, the combination erlotinib+bevacizumab totally prevented node invasion (P>0.05 vs controls) (P=0.007 vs controls). This RT-related effect on tumour cell proliferation was diminished by the presence of erlotinib+bevacizumab to a level similar to the controls .Neither erlotinib  nor bevacizumab  administered alone had a significant impact on tumour cell proliferation (ontrols) . In contP<0.001) increase in tumour angiogenesis. Of note, this RT-related effect on tumour vessels was no longer observed when RT was combined with erlotinib and bevacizumab. The results of the examination of peripheral tumour vessels were found to be superimposable with those of the central part of the tumour (data not shown).Bevacizumab given alone induced a decrease in endothelial cell VEGFR2 labelling . NeverthAll targeted treatments were found to diminish VEGFR2 expression on tumour cells . The comThere are recent preclinical background  or chemoper se and that erlotinib exhibited moderate anti-tumour effects as a single drug (Doses of individual agents were chosen to generate relatively limited effects and thus allow detection of potential interactions. This is for this reason that bevacizumab had no evident impact on tumour growth gle drug . Interesgle drug .As recently underlined by This study was also specifically designed to examine the impact of treatment not only on the primary tumour itself but also on the local metastatic invasion in nodes, thus creating a condition of clinical relevance. This was made possible by the adoption of a head and neck orthotopic model . Of clinIn total, this study provides another strong rationale for a combination between irradiation and EGFR pathway targeting plus anti-angiogenic treatment  in which


The following sentences were omitted from the Funding information: Additional support was provided by grant P50-GM076547 from the National Institute of General Medical Sciences. IS acknowledges support from the National Institutes of Health grant R01-GM072855.


The animals were sacrificed at day 24 after tumour implantation. IL-2 and histamine in combination significantly reduced tumour growth. The microvessel density was significantly reduced, an effect mainly affecting the small vessels. No obvious alteration in the pattern of VEGF mRNA expression was evident and no significant changes in apoptosis were observed. Neither IL-2 nor histamine alone caused any detectable effects on tumour growth. Histamine caused an early and pronounced decline in tumour blood flow compared to normal brain. The results indicate that the novel combination of IL-2 and histamine can be of value in reducing intracerebral tumour growth and, thus, it might be of interest to re-evaluate the therapeutic potential of biotherapy in malignant glioma. © 2000 Cancer Research CampaignBiotherapy including interleukin-2 (IL-2) treatment seems to be more effective outside the central nervous system when compared to the effects obtained when the same tumour is located intracerebrally. Recently published studies suggest that reduced activity of NK cells in tumour tissue can be increased by histamine. The present study was designed to determine whether IL-2 and histamine, alone or in combination, can induce anti-tumour effects in an orthotopic rat glioma model. One group of rats was treated with histamine alone (4 mg kg


The relationship between DNA double-strand break rejoining rates, inherent radiation sensitivity and tumour response to radiation therapy was determined for a group of 25 squamous cell carcinoma (SCC) and eight sarcoma (SAR) tumours. DNA double-strand break frequencies were measured by neutral filter elution in first passage following explant tumour samples after in vitro exposure to 100 Gy of 60Co gamma-rays. There was no significant difference between SCC and SAR tumour cells in their sensitivity to break induction, but in a 1 h time period SAR tumour cells rejoined significantly fewer breaks than SCC tumour cells, consistent with the greater sensitivity of SAR and suggesting that differences in rates of break rejoining account for the different distributions of radiosensitivities seen when different tumour types are compared. The percentage of breaks rejoined in 1 h in these tumour samples correlated well with D(o) and with the beta component of the survival curve, measured in vitro by clonogenic assay in tumour cell lines established from the tumour samples, but not with SF2 or the alpha component of the survival curve. The rates of DNA double-strand break rejoining therefore appear to influence the exponential portion of survival curves and probably the interactions between breaks. The percentage of breaks rejoined in 1 h was higher in SCC tumours that subsequently failed radiotherapy and, although the differences were not significant, they suggest that rates of break rejoining are an important component of tumour response to radiation therapy.


The relationship between tumour volume response and cell kill in B16 melanoma following treatment in vivo with cyclophosphamide (CY) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) was investigated. Tumour volume response, expressed as growth delay, was estimated from measurements of tumour dimensions. Depression of in vitro colony-forming ability of cells from treated tumours was used as the measure of tumour cell kill. The relationship between these parameters was clearly different for the two agents studied. CY produced more growth delay (7.5 days) per decade of tumour cell kill than CCNU (2 to 3.5 days). The possibility that this was due to a technical artefact was rejected in favour of an alternative explanation that different rates of cellular repopulation in tumours treated with CY and CCNU might be responsible. Cellular repopulation was measured directly, by performing cell-survival assays at various times after treatment with doses of CY and CCNU which produced about 3 decades of cell kill. The rate of repopulation by clonogenic cells was much slower after treatment with CY than with CCNU, and this appears to account for the longer duration of the growth delay obtained with CY.


Regional and distant lymph node cells, thoracic duct cells and peripheral blood lymphocytes from rats bearing a spontaneously metastasizing and apparently non-immunogenic sarcoma were assayed for cytotoxic activity on microcultures of tumour cells at 7, 14 and 21 days of tumour growth. In the regional lymph nodes detectable cytotoxicity was present at 7 days and the overall activity remained constant at 14 and 21 days. At Day 7 of tumour growth the cytotoxic cell population in the regional node was tumour specific in its cytotoxic effect, very radiosensitive and could not be removed by nylon wool column purification. In contrast the cells in the regional nodes at Day 21 were nonspecifically cytotoxic and could be completely removed by nylon wool treatment. In the peripheral blood, cytotoxic lymphoid cells not removed by nylon wool, were detectable at all stages of tumour growth. The thoracic duct lymph cells were, however, without cytotoxic activity throughout the period of tumour growth studied. Distant lymph node cells were assayed for cytotoxicity and it was found that they acquired significant cytocidal properties only late in tumour growth. The sera from tumour-bearing rats were tested for inhibitory activity on the cytotoxicity of Day 7 regional lymph nodes from tumour-bearing rats. It was found that a specific inhibitor appeared in the serum and that its activity increased with tumour growth. The possible contributions of the changes in lymph node cytotoxicity and the development of specific serum inhibitors to continued growth and dissemination of the tumour are discussed.


The tumours were ductal adenocarcinomas. Larger tumours showed invasive growth and spreading into the surrounding tissues, mainly into spleen and peritoneum. Microangiography revealed that the pancreatic tumours had an irregular and scanty vessel network and there were avascular areas in the center of the tumour. The area between normal pancreas and the induced tumour had dense vascularization. Intrapancreatic tumour induction with cultured pancreatic carcinoma cells produced a solid and uniformly growing tumour in Lewis rats and it thus provides a possible model for pancreatic cancer studies. © 2000 Cancer Research CampaignPancreatic cancer is one of the most frustrating problems in gastroenterological surgery, since there is little we can do to improve the survival of patients with current treatment strategies. If one is to elucidate factors related to carcinogenesis, tumour biology, diagnostics and new treatment modalities of this malignant disease, then it is essential to develop a suitable animal model. In the present study we investigated rat pancreatic tumour growth after intrapancreatic injection of cultured pancreatic carcinoma cells (DSL-6A/C1), originally derived from an azaserine-induced tumour, as well as the features of tumour microcirculation using the microangiography technique. After intrapancreatic inoculation, tumours were detected in 64% of animals. A 1 cm


We observed growing tumour vessels in vivo to study the relationship between blood flow and vascular enlargement. Mammary adenocarcinoma was implanted into Fisher-344 rat with dorsal skin-fold transparent chambers. Vascular growth was observed and recorded on videotape through a microscope for 6 h. Vascular networks were photographed and traced every 30 min to identify changes over time. Tumour sections were stained with Masson's trichrome and anti-Factor VIII-related antigen. Tumour growth was rapid enough for differences to be seen each hour. Vessels with a high blood flow showed an increase in diameter within a few hours and new branches formed from these vessels. In contrast, vessels without an increase in blood flow showed no change in diameter. Vessels within the interstitium surrounding the tumour were lined by endothelium that was positive for anti-Factor VIII-related antigen staining. Vessels in the tumour had extremely rare endothelial cells detectable by Masson's trichrome or anti-Factor VIII-related antigen staining. In conclusion, increased blood flow may cause vascular enlargement and some primitive vessels seem to lack endothelium.


We studied whether fibronectin (FN) enhances the activity of autologous tumour-reactive cytotoxic T lymphocytes (CTLs) generated from cancer patients. The proliferation of CTLs stimulated by immobilised anti-CD3 monoclonal antibody and interleukin 2 (IL-2) was enhanced three or four times by immobilised FN. whereas soluble FN did not alter the DNA synthesis of CTLs. Moreover, the cytotoxic activity of CTLs was augmented by FN stimulation against autologous tumour cells . The major cell surface phenotype of CTLs with FN was CD3+, CD4+ and CD25+ in 6 weeks' culture. Cytotoxicity against autologous tumour cells was inhibited by anti-HLA class I monoclonal antibody (MAb). The autologous tumour-killing activity of CTLs was suppressed by the elimination of CD4+ cells. Moreover, the cytokine production of CTLs was augmented by FN stimulation. Especially, the production of IL-2, interferon gamma (IFN-gamma), and granulocyte macrophage colony-stimulating factor (GM-CSF) was significantly augmented by FN stimulation (P<0.05). Thus, CTLs generated by FN might have both killer and helper functions, since they could lyse autologous tumour cells and secrete various cytokines, including IL-2.


Two rapidly growing allogeneic tumours, sublines of Yoshida (Y-P388) and Walker (W-256) injected intravenously in single cell suspensions produced tumour macrocolonies in the lungs of rats within 7 days. Y-P388 produced similar but fewer colonies in the kidneys. Colony forming efficiency (CFE) in lung was high in weanling rats given either sublethal whole body irradiation (WBI) or a single dose of rabbit anti-rat lymphocytic serum (ALS) to suppress immunity. In immunologically intact weanlings CFE was much lower and many 7-day old colonies showed signs of regression. CFE for primary tumour cell challenges decreased rapidly and markedly with increase in age of host during the first 1-2 weeks after weaning. This resistance to growth of a primary challenge in lungs of older rats was not significantly reduced by WBI but was decreased by ALS. CFE of a secondary challenge of tumour cells injected intravenously in rats which had been previously immunized with heavily irradiated (HR) tumour cells was very low; it was not significantly increased by WBI but was moderately increased by ALS. In weanling rats given lethal (900 rad) WBI, 1 hour before intravenous injection of tumour cells, treatment with bone marrow (BM) cells derived from normal adult donors increased CFE, whereas BM (or spleen) cells from immunized donors decreased CFE. The results suggest that ALS and WBI not only increase tumour CFE by suppressing immunity to tumour growth but also “condition” host tissue (tumour bed) in such a way as to facilitate the survival, “take” and initial replication of grafted tumour cells before the rats recover from the immunosuppressive effects of these treatments.


One explanation for the clinical association between tumour vascularity and probability of metastasis is that increased primary tumour vascularity enhances haematogenous dissemination by offering greater opportunity for tumour cell invasion into the circulation (intravasation). We devised an experimental tumour metastasis model that allowed manipulation of primary tumour vascularity with differential exposure of the primary and metastatic tumour site to angiogenic agents. We used this model to assess the effects of local and systemic increases in the level of the angiogenic agent basic fibroblast growth factor on metastasis. BDIX rats with implanted hind limb K12/TR adenocarcinoma tumours received either intratumoural or systemic, basic fibroblast growth factor or saline infusion. Both intratumoural and systemic basic fibroblast growth factor infusion resulted in significant increases in tumour vascularity, blood flow and growth, but not lung metastasis, compared with saline-infused controls. Raised basic fibroblast growth factor levels and increase in primary tumour vascularity did not increase metastasis. The clinical association between tumour vascularity and metastasis is most likely to arise from a metastatic tumour genotype that links increased tumour vascularity with greater metastatic potential.British Journal of Cancer (2002) 86, 123–129. DOI: 10.1038/sj/bjc/6600020www.bjcancer.comThe Cancer Research Campaign© 2002  Clinical studies of breast, lung, cervical and colorectal cancer suggest that tumour vascularity is related to risk of metastasis . One cauIn support of this, a relationship between tumour vascularity and the number of circulating tumour cells (CTC) has been demonstrated in an animal tumour model  – and thWe developed an experimental tumour metastasis model that allowed differential exposure of the primary and metastatic tumour site to the angiogenic agent basic fibroblast growth factor (bFGF), in order to vary primary tumour vascularity without altering tumour genotype. We assessed the effects of variation in tumour vascularity by both local and systemically administered bFGF, on tumour metastasis.The K12/TR cell line  is a transplantable hypovascular rat colonic adenocarcinoma that metastasises to the lungs from the subcutaneous implantation site  and a negative  control, were used. The cells were incubated for a further 4 days, and an MTT -2,5-diphenyltetrazolium bromide) assay then used to quantify cell number. MTT is a soluble tetrazolium salt that is converted by viable cells to an insoluble formazan precipitate  for 28 days from the time of cell injection. The bFGF was delivered at a dose rate of 100 ng per 24 h, as this dose has previously been shown to stimulate angiogenesis in a rat subcutaneous sponge model  that was placed in a subcutaneous pocket. The tumour cell inoculum was then continuously infused at a 0.25 μl h−1). bFGF was delivered at a dose rate of 5 μg per 24 h. This dose has previously been demonstrated to increase liver metastasis vascularity when infused systemically in a rat fibrosarcoma model .The tissue equilibration method  was used−1) that exceed those reported  autoradiography film with calibration standards and then exposed for 3 weeks. Autoradiographic images were digitized for analysis using a PC based image processing system , with a pixel resolution of 34 × 38 μm. Line profiles were drawn from the tumour edge to the tumour centre and the transmitted light intensity value of each pixel on the profile was recorded. Each film was calibrated using co-exposed I-125 standards and the light intensity values were converted to (I-125)-IAP concentrations, and then normalized with respect to the tumour edge. Between 6–8 line profiles drawn at intervals around each tumour boundary were averaged per tumour. Tissue concentration of (I-125)-IAP was used as a relative measure of blood flow. We have previously found (unpublished observations) that tissue blood flow is directly proportional to Iodoantipyrine concentration up to flow rates  sections were cut and fixed in acetone, and endogenous peroxidase activity was then blocked with 0.1% Hydrogen Peroxide. After washing in TRIS-buffered saline and blocking for non-specific activity with diluted normal rabbit serum , sections were incubated with monoclonal Ox-43 primary antibody  at a dilution of 1 : 300 for 30 min. Subsequent processing was carried out using the streptavidin-biotin method (Dako) after washing in TBS containing 1 ml 1% 10-ethyl ether. The complex was visualized using diaminobenzydine and counterstained with Mayer's Haemotoxylin. Positive and negative controls were respectively, rat heart muscle, and flank tumour without primary antibody exposure.After staining, tumour sections were independently assessed for vascularity by two of the authors (MM Davies and P Mathur) at × 400 magnification  without knowledge of infusion group. Vessel volume fraction was assessed by Chalkley's method  using a Proliferation was assessed by quantifying the expression of the 36 kDa nuclear protein Proliferating Cell Nuclear Antigen (PCNA). PCNA is an auxiliary protein for DNA polymerase and it accumulates from stages G1 to M of the cell cycle, its presence within a cell indicates that the cell is proliferating , the sections were incubated with the monoclonal primary anti-PCNA antibody  at a dilution of 1 : 100 for 30 min. The secondary and tertiary layers were applied as per the Dako streptavidin-biotin complex after washing in TBS containing 1 ml 1% 10-ethyl ether. The complex was visualized using diaminobenzydine and counterstained with Mayer's Haemotoxylin. Sections were examined at × 400 magnification using a Nikon (Optiphot) microscope  with a 245 times; 175 mm rectangular field placed in the eyepiece. One thousand cells were counted in eight randomly placed fields. All brown staining cells were considered positive. PCNA was quantified by the PCNA index which was defined as the percentage of positive staining cells per 1000 cells counted (2×b) , two perpendicular  measurements were made of the shortest and longest tumour dimensions. A value for the volume of each tumour was then derived according to the formula: Vol=(π/6)×(aAfter a 4-week primary tumour growth period and a further 8 weeks following flank tumour excision (see above), animals were killed, weighed and examined for lung metastases. Studies in mice suggest that macroscopic examination of lungs for the presence of metastases correlates closely with results of serial sectioning and microscopic examination . MacroscThe lungs of each animal were excised and fixed in methacarn (methanol-inhibisitol-acetic acid in the ratio 6 : 3 : 1) overnight and then examined using a dissecting microscope  for the presence of metastases and weighed. Brain, intra-abdominal organs, and axillary and inguinal lymph node groups were also macroscopically examined for metastases.U test. Repeated measures analysis of variance was used to assess vessel length density and tumour:tumour edge 125I (blood flow) ratio differences from tumour edge to centre between bFGF and saline infusion groups. Lung metastasis experiments were designed to provide an 80% power of detecting as significant (P<0.05), a 50% increase in lung metastasis with the bFGF-induced increase in flank tumour vascularity. This required a total sample of 28 animals in each metastasis experiment.Differences between bFGF and saline infused groups were assessed by Mann Whitney-All experiments using laboratory animals were carried out according to UKCCCR guidelines and were approved under the terms of the UK Home Office Animals (Scientific Procedures) Act 1986. All laboratory animal procedures had also been reviewed by the Royal Marsden Hospital Ethics Committee−1  and 30 ng ml−1  compared with control . Proliferation of cells exposed to bFGF at a concentration of 150 ng ml−1  was not significantly different (P=0.1) to control  in animal weights in bFGF (371 g (362–421 g)) compared with saline (392 g (362–428 g)) groups.Ten tumours  from 10 animals were examined. There was no difference (P=0.008) in vessel length density in the bFGF  compared with the saline  groups. Similarly, there was a significant increase (P=0.004) in vessel volume fraction in the bFGF  compared with the saline  group. There was also a significant increase (P=0.0001) in vessel length density with distance from the tumour edge between bFGF and saline infused tumours  between bFGF infused compared with saline-infused tumours (There was a significant increase (P=0.03) in PCNA index in the bFGF  compared with the saline  group. This was associated with a significant increase (P=0.05) in the volume of bFGF compared with saline infused tumours . There was no significant difference in animal weights (P=0.2) between bFGF (421 g (309–432 g)) and saline groups. There was also no significant difference in lung weight  in animal weights between bFGF (329 g (309–421 g)) and saline (319 g (298–398 g)) groups.Ten tumours  from 10 animals were examined. There was no significant difference (P=0.009) in vessel length density in the bFGF  compared with the saline-infused  group. Similarly, there was a significant increase (P=0.009) in vessel volume fraction in the bFGF  compared with the saline-infused  group. There was a significant increase (P<0.0001) in vessel length density with distance from the tumour edge between bFGF and saline-infused tumours  with distance from the tumour edge between bFGF and saline-infused tumours (There was a significant increase (P=0.04) in the bFGF  compared with the saline-infused  group. There was a trend that did not reach statistical significance (P=0.08), for an increase in tumour volume of bFGF compared with saline-infused tumours . There was no significant difference (P=0.4) in animal weights between bFGF (421 g (313–439 g)) and saline (413 g (304–459 g)) groups. There was also no significant difference in lung weight (P=0.2, Figure 7P=0.9, Twenty-eight animals were studied (systemic saline infusion in vitro K12/TR adenocarcinoma proliferation suggests that K12/TR proliferation was sensitive to bFGF. This is likely to have been mediated by bFGF receptors that have been described on the cell surface of some colonic adenocarcinomas (in vivo increases in flank tumour proliferation and growth that occurred with bFGF infusion.The finding that bFGF increased in vitro bFGF or grow more rapidly in vivo on exposure to bFGF (Intratumoural and systemic bFGF infusion increase tumour vascularity and blood flow in both the HSN sarcoma and the K12/TR adenocarcinoma cell lines ( to bFGF .Interstitial bFGF infusion approximately doubled K12/TR flank tumour vascularity, blood flow, proliferation and growth. These increases were compatible with the primary tumour vascularity increases that have been clinically associated with an increased risk of metastasis . It was Systemic bFGF infusion produced increases in flank tumour vascularity, blood flow and growth that were of a similar magnitude to those achieved with interstitial infusion of a 50-fold smaller bFGF dose. Thus systemically-administered bFGF was active at the flank tumour site. Despite systemic bFGF levels that were capable of increasing flank tumour vascularity and can be expected also to have reached the lungs, there was no significant increase in lung metastasis.These results do not support a role for raised levels of bFGF at either the primary or metastatic tumour site, or for associated increases in flank tumour vascularity and blood flow, in increasing metastasis. Even if increased primary tumour vascularity increased vascular intravasation . These s


The aim of this study was to examine the ability of AQ4N to potentiate the anti-tumour effect of cisplatin and to compare it to the chemopotentiation effect of tirapazamine. In the T50/80 murine tumour model, AQ4N (50–100 mg/kg) was administered 30 min, 2.5 or 6 h prior to cisplatin (4 mg/kg or 8 mg/kg); this produced an anti-tumour effect that was approximately 1.5 to 2 times greater than that achieved by a single 4 or 8 mg/kg dose of cisplatin. Tirapazamine (25 mg/kg) administered 2.5 h prior to cisplatin (4 mg/kg) resulted in a small increase in anti-tumour efficacy. AQ4N was also successful in enhancing the anti-tumour effect of cisplatin in the SCCVII and RIF-1 murine tumour models. This resulted in an increased cell kill of greater than 3 logs in both models; this was a greater cell kill than that observed for tirapazamine with cisplatin. Combination of cisplatin with AQ4N or tirapazamine resulted in no additional bone marrow toxicity compared to cisplatin administered alone. In conclusion, AQ4N has the potential to improve the clinical efficacy of cisplatin. © 2001 Cancer Research Campaign


N -[4-doxorubicin- N -carbonyl (oxymethyl) phenyl] O -β-glucuronyl carbamate (DOX-GA3) was synthesised for specific activation by human β-glucuronidase, which is released in necrotic areas of tumour lesions. This novel prodrug was completely activated to the parent drug by human β-glucuronidase with V max= 25.0 μmol min–1mg–1and K m= 1100 μM. The pharmacokinetics and distribution of DOX-GA3 in nude mice bearing human ovarian cancer xenografts (OVCAR-3) were determined and compared with DOX. Administration of DOX at 8 mg kg–1i.v.  to OVCAR-3-bearing mice resulted in a peak plasma concentration of the drug of 16.4 μM (t = 1 min). A 7.6-times lower peak plasma concentration of DOX was measured after injection of DOX-GA3 at 250 mg kg–1i.v. (50% of MTD). In normal tissues the prodrug showed peak DOX concentrations that were up to 5-fold (heart) lower than those found after DOX administration. DOX-GA3 activation by β-glucuronidase in the tumour yielded an almost 5-fold higher DOX peak concentration of 9.57 nmol g–1(P< 0.05) than the peak concentration of only 2.14 nmol g–1observed after DOX. As a consequence, the area under the curve of DOX calculated in tumour tissue after DOX-GA3 (13.1 μmol min–1g–1) was 10-fold higher than after DOX (1.31 μmol min–1g–1). The anti-tumour effects of DOX-GA3 and DOX were compared at equitoxic doses in OVCAR-3 xenografts at a mean tumour size of 125 mm3. The prodrug given i.v. at 500 mg kg–1weekly × 2 resulted in a maximum tumour growth inhibition of 87%, while the standard treatment with DOX at a dose of 8 mg kg–1i.v. weekly × 2 resulted in a maximum tumour growth inhibition of only 56%. Treatment with DOX-GA3 was also given to mice with larger tumours containing more necrosis. For tumours with a mean size of 400 mm3the specific growth delay by DOX-GA3 increased from 2.7 to 3.9. Our data indicate that DOX-GA3 is more effective than DOX and suggest that the prodrug will be specifically advantageous for treatment of advanced disease. © 2001 Cancer Research Campaign http://www.bjcancer.comThe doxorubicin (DOX) prodrug


The expression of bcl-2 was studied in normal ovaries and in ovarian tumours by immunohistochemical analysis. Normal epithelium was strongly stained in all nine examined ovaries. In comparison, all tumour groups showed a substantially decreased tumour cell expression of the same order of magnitude. Thus, benign tumour cells were weakly stained in two and unstained in two samples, while the remaining eight showed strong expression. Of ten borderline samples, one was unstained and five had weakly and four strongly bcl-2 positive tumour cells. Finally, 24 of 50 malignant tumours showed strong staining, while weak or no expression in tumour cells was found in 16 and 10 samples respectively. The reduced staining deviated significantly from normal ovary for both borderline (P = 0.02) and malignant groups (P = 0.01). Tumour cell staining with the bcl-2 antibody was significantly reduced when tumour mass had to be left behind compared with those with no visible remaining tumour (P = 0.03 and 0.003 for weakly and strongly stained tumours respectively). The expression of bcl-2 in malignant tumour cells was inversely correlated with the expression of p53. Bcl-2 expression was correlated with survival with significantly reduced survival in weakly (P = 0.02) and unstained (P < 0.001) groups compared with those patients having strongly stained malignant tumour cells. This correlation between the presence of bcl-2 and survival was maintained in the subgroups of patients with advanced disease or with residual tumour bulk and was also the case in patients having p53-positive tumours. Our results indicate an inhibitory role of bcl-2 in development and progression of ovarian tumours.


Here, we investigated whether the degree of tumour angiogenesis can be assessed Mice were xenografted with moderately  or highly vascularized  tumours and scanned up to 24 hours after intravenous SIDAG injection using fluorescence reflectance imaging. Contrast-to-noise ratio was calculated for all tumours, and fluorochrome accumulation was quantified using fluorescence-mediated tomography. The vascular volume fraction of the xenografts, serving as a surrogate marker for angiogenesis, was measured using magnetic resonance imaging, and blood vessel profile (BVP) density and vascular endothelial growth factor expression were determined.P < 0.0001), followed by HT1080, MCF7 and DU4475 tumours. Fluorescence-mediated tomography revealed 4.6-fold higher fluorochrome concentrations in MDA-MB435 than in DU4475 tumours . The vascular volume fraction was 4.5-fold , blood vessel profile density 5-fold (399 ± 36 BVPs/mm2 versus 78 ± 16 BVPs/mm2) and vascular endothelial growth factor expression 4-fold higher for MDA-MB435 than for DU4475 tumours.SIDAG accumulation correlated well with angiogenic burden, with maximum contrast to noise ratio for MDA-MB435 (in vivo using planar or tomographic imaging technology. They may thus facilitate characterization of solid tumours.Our data suggest that perfusion-type cyanine dyes allow assessment of angiogenesis  Contrast-enhanced optical imaging is an emerging modality that may be used to detect and characterize solid tumours such as breast cancer ,2. OpticHence, other cyanine-based contrast agents have recently been developed that possess more favourable optical and pharmacokinetic properties. SIDAG  is among these recently synthesized derivatives of ICG that have improved photophysical and pharmacological characteristics . In the preliminary set of experiments at the beginning of the project, the pharmacokinetic properties of the fluorochrome and the optimal time window for animal scanning had to be determined. For this purpose, four animals bearing MDA-MB435 tumours were scanned repetitively up to 72 hours after probe injection . Based on these data, all other animals were scanned 3, 6 and 24 hours after injection. Because MCF7 cells are hormone dependent, an oestradiol pellet  was implanted subcutaneously 1 week before tumour inoculation. All tumours were allowed to grow to approximately 5 to 7 mm in diameter before imaging studies were performed. In order to assess whether fluorochrome accumulation in different xenografts can reliably be distinguished even in the same individuals, five animals were co-implanted with both MDA-MB435 and DU4475 tumours. A total of 43 tumours were examined and evaluated in this study.All primary cell lines  used in this study were obtained from the American Tissue Culture Collection . All cell lines were cultured in modified Eagle's medium, RPMI-1640, or Dulbecco's modified Eagle's medium cell media supplemented with 10% foetal calf serum and 1% penicillin/streptomycin . Cells were grown using routine procedures in a monolayer culture at 37°C in a 5% carbon dioxide humidified atmosphere. To grow and propagate the tumours in mice, 2 × 10For reflectance and tomographic imaging studies, the mice were anaesthetized by intraperitoneal administration of ketamine (125 mg/kg body weight) and xylazine (12.5 mg/kg body weight). The average time of anesthesia was approximately 20 to 30 minutes and animals were allowed to wake up between imaging sessions. Before injection of the contrast agent, an anatomical white light image was obtained. SIDAG was dissolved in saline to achieve a 0.2 mmol/l solution and injected into the tail vein at a dose of 2 μmol/kg body weight. All experiments were performed with approval of the Institutional Animal Care Committee.tumour – SImuscle)/SDnoise .The whole body multichannel small animal imager is a prototype reflectance imager and has been described elsewhere   system , which has been described in detail elsewhere . FMT imaFluorochrome concentration in the target tissue was then automatically calculated from the reconstructed images.Magnetic resonance imaging (MRI) was performed using a clinical 3 Tesla magnetic resonance system  using a custom-made solenoid coil for animal imaging with a diameter of 70 mm . Tumour-bearing animals (n = 4 for each xenograft) were chosen randomly from the each group and were anaesthetized by intraperitoneal injection of ketamine (125 mg/kg body weight) and xylazine (12.5 mg/kg body weight). For stable vascular access, a jugular vein catheter  was inserted . Vascula2 (BVP/mm2).Six samples from each tumour type were chosen randomly and prepared, and each was embedded and snap frozen at -70°C immediately after imaging. For each tumour sample, on average 12 sections were selected using a systematic random sampling principle, as was described by Gundersen and Jensen . Immunoh7 cells) was collected and purified by centrifugation . To precipitate and thus concentrate the protein, a twofold volume of cold acetone was added to each sample and maintained at -20°C for 16 hours, followed by centrifugation at 10,000 g at 4°C for 30 minutes. The protein pellets were dissolved in 100 μl of 6× SDS sample buffer . Proteins were then electrophoretically separated in nonreducing 12% SDS-PAGE and transferred to polyvinylidene fluoride membranes . After blocking, immunoblots were incubated with VEGF polyclonal antibody  and then with a peroxidase-conjugated goat anti-rabbit IgG secondary antibody . Peroxidase activity was revealed using ECL chemiluminescence  and X-ray film, as described by the manufacturers. Recombinant human VEGF165  was used as a positive control. The developed films were scanned in an AlphaImager 2200  and the amount of protein was quantified using the provided Alpha Ease FC software (version 3.2.1). For comparison, the VEGF protein expression level in MDA-MB435 cells was set at 100%, and all other cell types were scaled accordingly.Secreted vascular endothelial growth factor (VEGF) was analyzed in the supernatant of cultivated tumour cells that had been maintained in serum-free medium for 24 to 48 hours . The supP < 0.05 was regarded to indicate statistically significant differences.Data are presented as mean ± standard deviation. Differences between the tumour xenografts  were statistically evaluated by analysis of variance with Bonferroni correction for multiple comparisons. Sequential FRI during the first 90 minutes after intravenous administration of contrast agent yielded moderate CNRs in the tumour tissue, even in highly vascularized tumours such as MDA-MB435 xenografts Figure . The tumP < 0.0001; Figure The results of all measurements are summarized in Table Co-implantation experiments with mice bearing both the highly vascularized MDA-MB435 and poorly vascularized DU4475 tumours (n = 5) confirmed the previous FRI findings and allowed clear differentiation of MDA-MB435 melanomas from DU4475 adenocarcinomas within the same animal  in tumour tissue for MDA-MB435 (3.58 ± 0.9%) than for DU4475  and MCF7  were also significantly lower than for MDA-MB435 xenografts.The independent 2), followed by the HT1080 tumours (128 ± 38 BVPs/mm2). A much lower BVPD was measured in the DU4475 (78 ± 16 BVPs/mm2) and MCF7 tumours  tissue, facilitating noninvasive probing of tissue with respect to oxyhaemoglobin and deoxyhaemoglobin, and fat and water content. Moreover, experimental data suggest that optical imaging is highly sensitive in resolving optical signatures, even in the picomolar range, which potentially can be exploited for molecular target detection in vivo .Recent clinical data with nonenhanced diffuse optical tomography (computed tomography laser mammography)  demonstrHowever, there is a good body of evidence that combining novel optical imaging technologies with fluorochrome-based optical contrast agents may markedly improve the sensitivity of the method, depending on fluorescent probe employed. In this regard, 'target-specific' or 'smart' fluorochromes can sensitively detect specific tumour epitopes, resolving structures even at picomolar concentrations, similar to scintigraphic imaging techniques.in vivo, the first clinical studies are currently being performed using non-target-specific, 'perfusion-type' fluorochromes, which are comparable to other clinically applied nonspecific contrast agents  [Although target-specific and 'smart' fluorochromes can be applied to resolve molecular targets  agents) . It remaThe purpose of this study was therefore to apply SIDAG, a new generation, 'perfusion-type' fluorochrome, for two-dimensional and three-dimensional optical imaging in human cancer xenografts that significantly differ in their degree of tumour angiogenesis.in vivo by iron oxide enhanced MRI as well as ex vivo by the BVPD and the level of VEGF expression as surrogate parameters of tumour angiogenesis. Planar and tomographic optical imaging revealed that retention of the fluorochrome in tumour xenografts made lesions clearly apparent. Interestingly, the retained fluorochrome concentration significantly increases with the degree of tumour angiogenesis, and both FMT and FRI data correlate with the VVF, BVPD and VEGF level. The data from this study therefore support the hypothesis that tumour-induced angiogenesis can indeed be noninvasively evaluated using a nonspecific, perfusion-type fluorochrome and appropriate imaging technology.Our data reveal significant differences in tumour vascularization of the xenografts, as measured The first clinical feasibility study examining ICG-enhanced diffuse optical mammography was that presented by Ntziachristos and coworkers . Those iThe use of ICG in breast imaging is restricted by many factors. Because of a propensity to bind to albumin 95%), ICG distributes predominantly in the vascular space and undergoes rapid uptake by the liver. The plasma level of ICG falls within minutes after intravenous administration, which shortens the period during which imaging can be done 5%, ICG d. FurtherIn the present study the visibility of tumour xenografts with FRI increased substantially over the first 3 hours, showing a more than sixfold increase in CNR. This phenomenon can be attributed to the fact that SIDAG extravasated through leaky tumour vessels and was retained within the tumour interstitium over the first hours, whereas nontarget structures (nontumourous tissue) exhibited marked washout subsequently, which resulted in an increase in CNR.The washin and washout phenomena described above are clearly depended on the tumour biology examined. Indeed, xenografts with a high degree of tumour angiogenesis (MDA-MB435 and HT1080) exhibited significantly higher SIDAG concentrations than did the poorly vascularized adenocarcinomas (MCF7 and DU4475).Generally, factors such as vessel density, capillary permeability inside the tumours, or a combination of both may be responsible for greater enhancement of tumour tissue, because the microvascular hyperpermeability observed in carcinomas increases with increasing tumour grade . This phCompared with planar imaging technology (FRI), the FMT data more closely resembled the data generated by iron oxide enhanced MRI, which is a well established tool for the assessment of tumour angiogenesis. Although, for example, MRI revealed an approximately 4.5-fold higher VVF for MDA-MB435 than for DU4475 tumours, FRI revealed only a 2.3-fold higher fluorescence signal. However, FMT resolved an approximately 4.6-fold higher fluorochrome accumulation in the MDA-MB435 tumours as compared with DU4475 adenocarcinomas. This may be attributed to ability to resolve and differentiate deep-seated tissue fluorescence (such as that deriving from the liver) from more superficially located tumour-associated fluorochrome accumulation. Although FRI cannot differentiate between deep-seated strongly fluorescent structures and superficial fluorochrome accumulation, cross-sectional imaging with FMT can do so. Thus, scattered fluorescence from deep-seated structures can interfere significantly with imaging results with FRI as compared with FMT.Contrary to most other clinically available cross-sectional imaging modalities, FRI and FMT allowed us to visualize the fluorochrome in highly vascularized tumours even 24 hours after injection with reasonable CNR. Moreover, the fluorochrome was administered at a dose of only 2 μmol/kg, which is more than two orders of magnitude lower than for gadolinum-enhanced MRI studies. These results therefore underline the high sensitivity of this imaging approach as compared with MRI, for example. More recent developments in the design of optical contrast agents include targeting tumour-specific receptors or the design of activatable 'smart' fluorochromes that can further boost the CNR, given an appropriate tracer design ,27.It is clear that fluorescence-enhanced optical imaging not only has tomographic applications in breast cancer imaging but also applications for a variety of other tumour types. Experimental studies have focused on imaging dysplastic colonic polyps, lung tumours and intracerebral malignomas . MoreoveThe present study has a few limitations: It remains unclear whether SIDAG-enhanced FMT/FRI can resolve subtle changes in vascular density or tumour perfusion, caused by antianiogenic therapy. Moreover, we did not conduct pharmacokinetic studies to analyze dynamically the distribution of the fluorochrome between intravascular and extravascular compartments. However, these issues will be addressed in future projects.The results of the present study indicate that clinically relevant tissue parameters such as tumour angiogenesis can reliably be estimated using a perfusion-type optical contrast agent and planar or tomographic optical imaging methods. These types of agents are about to enter the clincal arena, and their use in diffuse optical tomography of the breast may facilitate lesion detection and characterization.BVP = blood vessel profile; BVPD = blood vessel profile density; CNBR = contrast-to-noise ratio; FMT = fluorescence mediated tomography; FRI = fluorescence reflectance imaging; MRI = magnetic resonance imaging; ICG = indocyanine green; ROI = region of interest; SIDAG = 1,1'-bis-(4-sulfobutyl)indotricarbocyanine-5,5'-dicarboxylic acid diglucamide monosodium; VEGF = vascular endothelial growth factor; VVF = vascular volume fraction.The authors declare that they have no competing interests.in vivo optical imaging experiments, analyzed the data and contributed to manuscript preparation. TP carried out the MRI experiments and participated in drafting the final manuscript. PH, KL, MS and SM developed the optical contrast agent, determined the tumour vessel density and contributed to drafting of the final manuscript. AvW carried out the western blotting. WH and AvW were involved in drafting the final manuscript.CB conceived the study, supervised the experiments and completed the final draft of the manuscript. AW and LM carried out the


We have correlated the morphological features of 30 human thyroid carcinomas with the cellular localisation of insulin-like growth factor 1 (IGF-1) mRNA and IGF-1 receptor peptide using in situ hybridisation with digoxigenin-labelled oligoprobes and immunohistochemistry. Four of the five follicular carcinomas studied showed a consistent, uniform, strong positivity for IGF-1 mRNA in tumour cells compared with weakly positive surrounding normal follicular tissue and negative stroma. The majority of papillary carcinomas showed weak to moderate epithelial positivity for IGF-1 mRNA and negative stroma. Immunohistochemistry for IGF-1 receptor showed moderate positivity confined to the tumour epithelial cells in both follicular and the majority of papillary carcinomas. However, in a subgroup of papillary carcinomas characterised by a diffuse stromal lymphoid infiltration (n = 5), the stromal cells showed a much stronger reactivity for IGF-1 mRNA than the tumour or background thyroid, and the tumour cells showed a uniformly high level of immunoreactivity for IGF-1 receptor. These results are compatible with the growth of the papillary carcinoma in these cases being the result of a symbiotic relationship between the stromal lymphoid cells and the tumour epithelium with the lymphoid cells responding to an antigen produced by the tumour cells and the tumour cells responding to a growth factor produced by the lymphoid infiltrate. We suggest that this mechanism may be important in other tumours regularly associated with a widespread lymphoid infiltrate.


Sir,The authors acknowledge the comments advanced by Dr Böhm. A major concern was about the seemingly disparate results presented in our studies . No suchγ-H2AX foci, to measure dsb repair in tumour cells.Two points can be extracted from this. (1) There are significant differences between normal and tumour cells in the variation of dsb induction. (2) Standard gel electrophoresis is suitable to determine dsb induction in both tumour and normal cells, but repair capacity can only be determined in normal cells (fibroblasts). This, however, does not mean that the dsb repair capacity is not relevant for the cellular radiosensitivity of tumour cells. To our best knowledge, there are no appropriate methods at hand, for several reasons not even recording of The differences in dsb induction among tumour cell lines is likely to be explained by variation in their chromatin structure and thus in their protective capacity from DNA damage, while normal cells maintain a constant chromatin structure. This is supported by many recent results emphasising variant patterns of chromatin methylation and histone acetylation in tumour cells. Moreover, the p53 status which is described in a second paper determine the cellular response to radiation, whereby micronuclei and apoptosis, as brought forward by Dr Bohm, may well be the consequence of dsb.


Since the tumour-selective cytotoxic activity of activated macrophages in vitro can be attributed to depletion of the culture medium of L-arginine by macrophage arginase, a series of experiments was designed to determine whether such a mechanism could operate in vivo. Extracellular fluid obtained from Gullino chambers within established tumours contained high levels of arginase, no detectable arginine and high levels of ornithine. When tumours were disaggregated into single-cell suspensions, arginase was readily detected within tumour macrophages but not within malignant cells. Inflammatory ascites induced in mice by Corynebacterium parvum was rich in arginase, depleted of L-arginine and cytotoxic in vitro to L5178Y and V79 cells. High levels of arginase in the ascites fluid were associated with resistance to challenge with syngeneic L5178Y cells. Lymph collected from the cisterna chyli in rats bearing a macrophage-rich sarcoma on the small bowel contained elevated levels of arginase, was depleted of arginine and contained increased concentrations of ornithine. We conclude that in sites of macrophage infiltration there is microenvironmental arginine depletion due to the action of arginase, and that arginase release could represent an important macrophage effector mechanism against a variety of targets, including malignant cells, virus-infected cells, fungi and parasites.


Long-circulating  liposomes coated with polyethylene glycol (PEG), which show reduced uptake by the reticuloendothelial system (RES) and enhanced accumulation in tumours, were used for conjugation to monoclonal antibodies (MAbs) as a drug-targeting device. A MAb (N-12A5) directed against erbB-2 oncoprotein, a functional surface antigen, was used. Amplification and overexpression of the erbB-2 gene product, being unique to malignancy, confer onto this antibody-mediated therapy high tumour specificity. In vitro binding of [3H]cholesteryl ether ([3H]Chol ether) labelled anti-erbB-2 conjugated liposomes to N-87 cells (erbB-2-positive human gastric carcinoma) was compared with the binding of non-targeted liposomes and indicated a 16-fold increase in binding for the targeted liposomes. No difference in binding to OV1063 cells (erbB-2-negative human ovary carcinoma) was observed. These results indicate highly selective binding of antibody-targeted liposomes to erbB-2-overexpressing cells. Despite increased cell binding, doxorubicin (DOX) loaded in anti-erbB-2-conjugated liposomes did not cause increased in vitro cytotoxicity against N-87 cells, suggesting lack of liposome internalisation. In vivo, the critical factor needed to decrease the non-specific RES uptake and prolong the circulation time of antibody-conjugated liposomes is a low protein to phospholipid ratio ( < 60 micrograms mumol-1). Using these optimised liposome preparations loaded with DOX and by monitoring the drug levels and the [3H]Chol ether label, biodistribution studies in nude mice bearing subcutaneous implants of N-87 tumours were carried out. No significant differences in liver and spleen uptake between antibody-conjugated and plain liposomes were observed. Nevertheless, there was no enhancement of tumour liposome levels over plain liposomes. Both liposome preparations considerably enhanced DOX concentration in the tumour compared with free drug administration. Therapeutic experiments with N-87 tumour-bearing nude mice indicated that anti-tumour activity of targeted and non-targeted liposomes was similar, although both preparations had an increased therapeutic efficacy compared with the free drug. These studies suggest that efficacy is dependent on drug delivery to the tumour and that the rate-limiting factor of liposome accumulation in tumours is the liposome extravasation process, irrespective of liposome affinity or targeting to tumour cells.


The dual properties of genetic instability and clonal expansion allow the development of a tumour to occur in a microevolutionary fashion. A broad range of pressures are exerted upon a tumour during neoplastic development. Such pressures are responsible for the selection of adaptations which provide a growth or survival advantage to the tumour. The nature of such selective pressures is implied in the phenotype of tumours that have undergone selection. We have reviewed a range of immunologically relevant adaptations that are frequently exhibited by common tumours. Many of these have the potential to function as mechanisms of immune response evasion by the tumour. Thus, such adaptations provide evidence for both the existence of immune surveillance, and the concept of immune selection in neoplastic development. This line of reasoning is supported by experimental evidence from murine models of immune involvement in neoplastic development. The process of immune selection has serious implications for the development of clinical immunotherapeutic strategies and our understanding of current in vivo models of tumour immunotherapy. © 2000 Cancer Research Campaign


A comparison of the cytotoxic effectiveness of adriamycin incorporated into ion exchange microspheres with conventional chemotherapeutic use of adriamycin was carried out in a rat tumour model. Drug microspheres were targeted to the tumours by embolisation into the arterial supply of the hind limb bearing the tumour. Microspheres were found to embolise in tumour tissue at concentrations of up to 39 times that of the surrounding normal tissue. As a result, adriamycin microsphere therapy was found to retard significantly (P less than 0.01) tumour growth rates compared to growth rates associated with similar doses of adriamycin delivered as free drug rather than bound to controlled release microspheres. Equivalent sham microsphere treatments showed no significant difference in tumour growth rates compared with the control group. Adriamycin loaded on to ion exchange microspheres holds strong potential for treatment of human malignancy.


In a study based on formalin-fixed paraffin-embedded material from 86 patients with primary synovial sarcoma located in the extremities or on the trunk wall, the prognostic importance of MIB-1 index, p53-expession and tumour size was analysed. Multivariate analysis identified two metastatic risk factors: increasing tumour size and MIB-1 >9%. The 5-year metastasis-free survival-rate for patients with tumour size ≤5 cm + MIB-1 <10% was 0.83  0.64–0.92) compared to 0.31 (95% CI 0.11–0.53) in cases with tumour size >5 cm + MIB-1 ≥10%. Our study shows that metastatic disease in synovial sarcoma is closely related to MIB-1 index. Using our model based on tumour size and MIB-1 index, cases with good and poor prognosis can easily be discriminated. Therefore our model can be used to identify patients who should be considered for adjuvant chemotherapy. © 1999 Cancer Research Campaign


Competitive polymerase chain reaction assays have been developed for the quantitation of oestrogen receptor mRNA and two oestrogen-regulated mRNAs (progesterone receptor and pNR-2/pS2) in breast cancer cells. These assays are more sensitive than traditional hybridisation techniques, do not require the use of radioisotopes, measure absolute amounts of messenger RNAs and can be used to measure the expression of mRNAs in small numbers of tumour cells obtained by fine-needle aspiration (FNA). These assays should prove useful for predicting the hormone responsiveness of breast cancer from tumour cells obtained by FNA at diagnosis and could be particularly useful in the management of elderly/frail patients who receive primary tamoxifen, or in other patients for whom tumour tissue for standard biochemical measurements is not available.


P = 0.0005, P = 0.035 respectively) and local control  than those with radiosensitive and poorly vascular tumours. No significant correlation was seen between IMD and SF2. Multivariate analysis (including tumour stage and patient age) showed that only SF2 and IMD were significant prognostic factors for survival. Patients with both a radioresistant and highly vascular tumour had a 5-year survival level of 18% compared to 77% for those patients with a radiosensitive and poorly vascularized tumour. Tumour angiogenesis and cellular radiosensitivity are independent prognostic factors for cervix carcinoma treated with radiotherapy. Allowing for tumour radiosensitivity increases the prognostic significance of vascularity measurements in cervix tumours. © 1999 Cancer Research CampaignThe aim of the study was to investigate the relationship between intrinsic radiosensitivity and vascularity in carcinoma of the cervix given radiotherapy, and assess whether more refined prognostic information can be gained by combining the two parameters. A retrospective study was carried out on 74 patients with locally advanced carcinoma of the cervix. Formalin-fixed, paraffin-embedded tumour biopsies were stained with anti-factor VIII using immunohistochemistry. Vascularity was scored using the intra-tumour microvessel density (IMD), or ‘hot-spot’, technique. For the same patients, the measurement of intrinsic radiosensitivity (SF2) had been made previously on the same pretherapy samples. Patients were stratified by the median IMD and SF2 scores. Women with radioresistant and highly vascular tumours had poorer 5-year survival (


Lymphoid cells stimulated by soluble tumour antigens in the MCA-induced murine fibrosarcoma system have been identified by subclass and protective capacity in adoptive syngeneic hosts. Lymph-node or spleen cells taken at weekly intervals after inoculation of syngeneic chemically induced fibrosarcomas were enriched by 3 methods in T, B, and "null" cell subclasses, and assayed for proliferative kinetics in response to soluble membrane antigens. The stimulated subpopulations were found to be heterogeneous, their composition varying with time and tumour burden. Initial proliferative responses after tumour inoculation were limited to the T-enriched subpopulation. Later during tumour growth, T, B and null cell fractions were vigorously and equally stimulated by tumour antigen. The ability of the same T, B or null-cell subpopulations to inhibit tumour growth was measured in adoptive hosts by a modified Winn assay. Only the T-cell subpopulation responding to tumour antigen in vitro effectively and consistently retarded tumour growth in vivo. In contrast to the shared specificities on syngeneic tumours identified by the proliferative assay, tumour-growth inhibition was limited to the specific tumour borne by the cell donor.


The "chemosensitizing" properties of the radiosensitizer misonidazole (MISO) were examined in 2 tumour systems, murine Lewis lung carcinoma and human pancreatic adenocarcinoma xenografted into immune-suppressed mice, using a soft-agar colony assay to measure tumour-cell survival. In mice bearing Lewis lung tumour, the administration of MISO simultaneously with melphalan, cyclophosphamide. CCNU, FU or vincristine gave substantial enhancement of cytotoxicity (DEFs from 1.5 to 3.5). However, no enhancement was seen with bleomycin, VP 16-213 or cis-Pt. The same level of enhancement of cyclophosphamide effect (DEF = 2.0) was seen with both cell survival and growth delay end-points effect (DEF = 2.0) was seen with both cell survival and growth delay end-points of tumour response. Enhancement was also seen in the human tumour xenograft with melphalan, cyclophosphamide and MeCCNU, using a cell survival assay, but cis-Pt was again not enhanced.


Tumour blood vessels differ from their normal counterparts for reasons that have received little attention. We report here that they are of at least six distinct types, we describe how each forms, and, looking forward, encourage the targeting of tumour vessel subsets that have lost their vascular endothelial growth factor-A (VEGF-A) dependency and so are likely unresponsive to anti-VEGF-A therapies. It has been known for more than a century that tumours have their own blood supply and that, for the better part of that time, the tumour vasculature is highly abnormal. At one time it was thought that the tumour vasculature was actually superior to that of normal tissues; this misconception arose because tumour vessels are often of large size and were, therefore, more conspicuous than the smaller, more numerous and functionally more effective blood vessels of normal tissues. By the early 1970s, however, it was clear that the overall blood flow tended to be significantly lower in tumours than in normal tissues, and that tumours needed to induce a vascular supply if they were to grow beyond minimal size. It was also suspected that tumours induced their neovasculature by secreting angiogenic factors, but the identity of these factors was just beginning to be investigated. In the years that followed, much has been learned about the molecular basis of angiogenesis and particularly about the central importance of one angiogenic factor, that is vascular permeability factor/vascular endothelial growth factor . Subsequent work has elucidated the steps and mechanisms by which VEGF-A induces the formation of tumour blood vessels. Furthermore, recent successes with agents that block VEGF-A or its receptors have supported This minireview addresses several specific questions that are relevant to these basic issues: what are tumour vessels and in what respects do they differ from normal blood vessels? How do tumour vessels form? Why are they abnormal? And, finally, why might an understanding of tumour blood vessel diversity be important clinically? Hopefully, addressing these questions will clarify some of the mechanisms by which tumours induce their abnormal blood vessels and lead to the development of therapies that more effectively target them.Tumour blood vessels have often been regarded as if they were a single entity, ‘angiogenic blood vessels’. However, it has long been known that tumour blood vessels are heterogeneous with regard to organisation, function and structure . Whereas164 (Ad-VEGF-A164) have contributed importantly to our understanding of the mechanisms by which tumours generate new blood vessels. When injected into the tissues of immunodeficient mice, Ad-VEGF-A164 induces the formation of each of the different types of tumour blood vessels listed in 164 are largely independent of the tissue in which they arise; similar vessel types form with similar kinetics in a wide variety of normal mouse and rat tissues, including skin, subcutis, fat, skeletal and heart muscle, and brain (164 has a characteristic morphology and has been given the name ‘mother vessel’ (MV)  structures that do not allow microvessels to expand their cross-sectional area by more than ∼30%  are found not only in a wide variety of VEGF-A-expressing human tumours, particularly glioblastoma multiforme, but also in cancers of the stomach and breast, where they are associated with an unfavourable prognosis  by retaining their large size and acquiring a smooth muscle-cell coating. Vascular malformations are readily distinguished from normal arteries and veins by their inappropriately large size (for their location) and by their thinner and often asymmetric muscular coat. Vessels of this description closely resemble the non-malignant VMs that occur, for example, in the skin, brain, etc. . Vascula164 also stimulate abnormal arteriogenesis and venogenesis, leading to the formation of the large, often tortuous blood vessels that supply and drain the tumour microvasculature  are generated by the alternative splicing of a single gene . These iheparans . VEGF-A1120 develop to term but generate a capillary network with larger-sized vessels and fewer branch points , but with few, small intratumour vessels  clearly ‘provide different signalling outcomes, even though they act through the same cell surface receptor (VEGFR2)’  or its receptors have provided proof of principle that anti-angiogenesis can be a useful adjunct to tumour therapy . Second,Tumours induce their vasculature through the unbalanced, local overexpression of a small number of growth factors, particularly VEGF-A. Tumour neovascularization thus differs significantly from the sprouting angiogenesis of normal vascular development, a process that is as yet not well understood, but that results, at least in part, from tightly regulated VEGF-A gradients and the balanced expression of many other growth factors and inhibitors. Tumours generate at least six distinct types of new blood vessels that differ from each other, and from the normal vasculature, with respect to organisation, structure and function. This classification has potential clinical and therapeutic significance. Current anti-VEGF/VEGFR approaches more likely target only a subset of angiogenic tumour blood vessels, that is, MV and GMP, that are dependent on exogenous VEGF-A. By contrast, the mature, smooth muscle-cell-coated daughter blood vessels, as well as the large feeder arteries and draining veins that result from arteriogenesis and venogenesis, are unlikely to be targeted, as they have lost VEGF-A dependence. Feeder arteries and draining veins would seem to be particularly valuable targets, because they supply and drain all of the smaller, angiogenic vessels enclosed within the tumour mass. Finding drugable targets on these blood vessels will require them to be better characterized. However, the genomic and proteomic revolution should make it possible to find such targets, especially because (as success with anti-VEGF/VEGFR drugs has shown) differences between tumour and normal blood vessels need not be absolute for therapy to be effective.


Noradrenaline induced changes in the distribution of blood flow in implanted tumour and normal liver tissue was measured using blood flow tracer microspheres. The ratio of embolised microspheres in tumour compared to normal tissue was determined before and after the intravenous infusion of noradrenaline, propranolol and a combination of the two drugs. The ratio was significantly decreased by noradrenaline alone but significantly increased when propranolol was added to the infusate. Propranolol had no effect on the ratio. The drug combination increased the tumour to normal ratio by approximately 69% and also doubled the proportion of microspheres entering the internal tumour circulation. This represents an enhanced relative blood supply to tumour and would provide a means of preferential carriage of blood borne cytotoxic agents to tumour tissue rather than normal tissue.


Unrestrained growth of various malignant tumours has been shown to depend upon a critical number of tumour cells which have switched to the angiogenic phenotype. Angiogenic phenotypes were noted in the early stage of prostatic carcinoma (PCa). We investigated 65 cases of latent PCa to define the correlation between tumour angiogenesis and tumour volume. Tumour angiogenesis was determined by the blood capillary density ratio (BCDR) evaluated by a colour image analysis system. Using experimental regression analysis, the correlation between the BCDR and PCa volume was divisible into two distinct stages. When the PCa showed a volume of more than 83 mm3, there was a significant positive correlation between the BCDR and PCa volume (rS-test P < 0.001). However, when the PCa showed a volume of less than 83 mm3, the BCDR remained at a low level which did not change until larger volumes were present . The present study suggested that latent PCa showing a volume of less than 83 mm3 would be 'early' indolent carcinoma which, on undergoing additional events concerning tumour angiogenesis, would assume more aggressive growth.


The effect of exogenous sex hormones on the cell mediated response in male and female mice has been studied by measuring the subcutaneous growth of HeLa tumour nodules and the variation in the total lymphocyte count. It was found that oestrogen treated male and female mice experienced a profound lymphopenia which was vary rapid in onset. Concurrent with the lymphopenia there was prolongation of HeLa tumour nodule growth in female mice, but not in males. A lymphopenia occurred in androgen treated male mice with subsequent prolongation of HeLa tumour nodule growth, and a lymphocytosis in female mice, with reduction of HeLa tumour nodule growth.


This study, aimed to define its anticancer efficacy against a series of murine and human tumour models, has provided evidence of major antitumour activity for F 11782. This was demonstrated as a high level of activity against the P388 leukaemia, as reflected by increased survival of 143–457%, when administered i.p., p.o. or i.v. as single or multiple doses, and proved consistently superior to etoposide or camptothecin tested concurrently. Single or multiple i.p. doses of F 11782 also proved highly active against the s.c. grafted B16 melanoma, significantly increasing survival (P < 0.001) and inhibiting tumour growth (T/C of 0.3%), again superior to etoposide tested concurrently. Furthermore, F 11782 inhibited the number of pulmonary metastatic foci of the B16F10 melanoma by 99%. In human tumour xenograft studies, multiple i.p. doses of F 11782 resulted in major inhibitory activity against MX-1 (breast) tumours (T/C of 0.1%), as well as causing definite tumour regressions, whereas none resulted from similar experimental treatments with etoposide. Significant activity was also recorded with F 11782 against the relatively refractory LX-1 (lung) xenografts, with an optimal T/C value of 19%. It was notable that the antitumour activity of F 11782 was consistently demonstrated over a wide range of 2–6 dose levels, providing evidence of its good overall tolerance. In conclusion, these results emphasize the preclinical interest of this novel molecule and support its further preclinical development. © 2000 Cancer Research Campaign


Transurethral resection of the prostate gland with irrigation fluid at room temperature leads to perioperative hypothermia which could give rise to adverse cardiovascular events in the perioperative period. The use of isothermic irrigation fluid reduces but does not eliminate this risk. Routine use of warm intravenous fluids along with isothermic irrigation had not been documented. This study set out to investigate the effect of the use of warm intravenous fluid together with isothermic irrigation fluid on the body temperature in patients undergoing transurethral resection of the prostate gland.One hundred and twenty consented patients with obstructing benign prostatic hyperplasia were randomly assigned to one of 3 groups. Group 1 received irrigation and intravenous fluids at room temperature, group 2 received warmed irrigation fluid at 38°C along with intravenous fluid at room temperature while group 3 patients received warmed irrigation fluid and warmed intravenous fluids at 38°C. Their perioperative body temperature changes were monitored, analyzed and compared.The mean decrease in body temperature at the end of the procedure was significantly greater in group 1 (0.98 ± 0.56°C) than in group 2 (0.42 ± .21°C) (p < 0.001). Significantly more patients in group 1 also experienced shivering. However, in group 3, there was no significant change in the mean body temperature (p > 0.05) and none of them felt cold or shivered.It is concluded that the use of isothermic irrigation fluid together with warm intravenous fluids during TURP prevents the occurrence of perioperative hypothermia.CCT-NAPN-15944 Perioperative hypothermia is an unintentional drop in the core body temperature to less than 36°C during or immediately following a surgical operation . ContribIn 1995 when TURP under caudal block regional anesthesia with boiled water as irrigation fluid , was comBetween January 2001 and December 2005, a total of 120 patients scheduled for transurethral resection of the prostate gland at our institution were enrolled into this randomized study. The study was approved by the hospital ethical review committee and all 120 patients provided written informed consent. The patients were randomly assigned to one of 3 groups by picking a ballot. Group 1 received irrigation and intravenous fluids at room temperature, group 2 received warmed irrigation fluid at 38°C along with intravenous fluid at room temperature while group 3 patients received warmed irrigation fluid and warmed intravenous fluids at 38°C. Previously boiled water which was allowed to cool was the irrigation fluid used for all groups. For groups 2 and 3, the appropriate temperature was achieved by mixing previously boiled and cooled water with varying amounts of freshly boiled water. The intravenous fluid for group 3 was warmed by immersing 4 bags of 500 ml intravenous fluids in a bucket containing warm water at 45°C. Only normal saline was used as intravenous fluid. Patients with co-morbid conditions such as diabetes mellitus, inguinal hernia, vesical calculi, asthma, axial skeletal or hip deformities which interfered with Lloyd Davis positioning, or recent cerebrovascular accidents were excluded from the study.The temperature in the room where the intravenous fluid and irrigation fluids were stored was recorded on the days the fluids were needed for surgery. Each patient had his oral temperature monitored every 5 minutes during the procedure and immediately after prostatic resection, before he was taken out of the theatre. Their pulse rate, blood pressure and respiration were monitored. They were covered with theatre linen at room temperature during the procedure and were asked to indicate if they felt cold at any point during or after resection before their discharge. They were also observed for occurrence of shivering. If the patients in groups 1 and 2 complained of cold, they were externally warmed by covering them with warm blankets and commenced on warm intravenous infusion to achieve internal warming also. The duration of hospital stay after their surgery was also documented.The data was analyzed using the SPSS 11.0 for Windows. Comparison of means was carried-out using the Levene's statistical test and p-value ≤ 0.05 was considered as significant.One hundred and twenty patients were studied. Table The 3 groups of patients were matched for age, volume of the prostate gland, weight of the resected gland, volume of irrigation fluid used, resection time and the core body temperature at the commencement of the procedure and are therefore comparable table .The decrease in the mean core body temperature was significantly more in the patients who had resection with irrigation fluid and intravenous fluid at room temperature. More of them felt cold and subsequently shivered. These findings are in keeping with those from previous studies where significantly more patients developed perioperative hypothermia following the use of irrigation and intravenous fluids at room temperature during transurethral resection of their prostate gland ,8,10,11.We recommend that both the irrigation fluid and the intravenous fluids used during TURP should be isothermic. This will prevent the occurrence of perioperative hypothermia and may help reduce the occurrence of adverse cardiac events which contribute to the morbidity and mortality associated with TURP when irrigation fluid at room temperature is used.The author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:


The GER human pancreatic carcinoma cell line possesses receptors for 1,25-dihydroxyvitamin D3. We report that the vitamin D analogue EB 1089 inhibits the growth of these cells in vitro and when grown as tumour xenografts in immunodeficient mice. Tumour-bearing mice were given EB 1089 at a dose of 5 microg kg(-1) body weight i.p. thrice weekly for 4-6 weeks. Tumour growth was significantly inhibited in treated animals compared with controls in the absence of hypercalcaemia. These findings may have therapeutic implications in pancreatic cancer.