Dynamic contrast agent-enhanced magnetic resonance imaging measurements of the perfusion of an immunogenic murine tumour showed that immune rejection was preceded by an increase in the apparent vascular volume of the tumour. This increase in vascularity, which has been observed previously in other tumours undergoing immune rejection, was confirmed by histological analysis of tumour sections obtained postmortem. Magnetic resonance imaging measurements similar to this could be used in the clinic to monitor the early responses of tumours to immunotherapy, before there is any change in tumour growth rate or volume. The early detection of tumour responses to therapy will be valuable both in the development of new therapies and in their subsequent implementation in the clinic, where early response detection could be used to tailor therapy to individual patients.The most widely used criterion for tumour response has been tumour shrinkage, however with some therapies, and particularly during early stage clinical trials, tumours may show a positive response to therapy in the absence of any change in tumour volume. For example, the antivascular drug combretastatin can produce dramatic changes in tumour blood flow without significantly affecting tumour volume . Such mehttp://www.cancer.gov/clinical_trials).Tumour immunotherapy has, until recently, met with limited success. However, a better understanding of how cytotoxic T lymphocytes respond to tumour cells, the molecular signalling pathways that regulate responsiveness and the mechanisms by which tumours evade immune surveillance coupled with the identification of tumour antigens has led to renewed interest in this therapeutic approach . Indeed,We show here how dynamic contrast agent-enhanced MRI (DCE-MRI) measurements can be used to detect changes in the vascularity of tumours undergoing immune rejection, prior to a reduction in tumour volume. We suggest that similar measurements could be used clinically to detect the early response of tumours to immunotherapy.α, goat polyclonal antibodies recognising CD-68 (M-20) and a secondary anti-rabbit IgG staining system (‘Immuno-Cruz staining system’) were purchased from Santa Cruz Biotechnology Inc., USA. Rabbit anti-human Factor VIII antibody was purchased from DAKO, Copenhagen, Denmark. Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies recognising rabbit and goat IgGs were purchased from Sigma . All other reagents were of analytical grade.Rabbit antiovalbumin antibody was purchased from Research Diagnostics Inc., NJ, USA. Rabbit polyclonal antibodies recognising CD4, CD8-ML-glutamine, penicillin (100 units ml−1) and streptomycin (100 μg ml−1). Selection of E.G7-OVA cells was maintained using culture medium containing 400 μg ml−1 G418.The E.G7-OVA cell line was originally derived from the murine thymoma line, EL-4, by transfection with a neomycin-selectable vector expressing full-length chicken ovalbumin (6) were injected subcutaneously into the shaved flanks of mice. Tumour size was measured using calipers and is reported as the product of the two largest perpendicular diameters (mm2). All experiments were conducted in compliance with a project licence issued under the Animals (Scientific Procedures) Act 1986 and were designed with reference to the UKCCCR guidelines for the welfare of animals in experimental neoplasia. The work was approved by a local ethical review committee.Female C57BL/6 mice were purchased at 6–8 weeks of age from Charles River Ltd (UK). Tumour cells  as the chromogen. As a negative control, the sections were treated with nonimmune rabbit serum. For immunofluorescence staining, the slides were prepared as described for immunoperoxidase staining, except that the peroxidase-block step was omitted. Slides were incubated with an appropriately diluted primary antibody before detection with a fluorescein-conjugated secondary antibody.Tumours were fixed in 10% formalin and embedded in paraffin. Five-micrometre thick sections were cut and stained with haematoxylin and eosin (H&E) or Masson's trichrome stain (Sigma). For immunoperoxidase staining, the rabbit primary antibodies were detected using a biotinylated anti-rabbit IgG secondary antibody and horseradish peroxidase (HRP)-conjugated to streptavidin using the Immuno-Cruz staining system. The formalin-fixed and paraffin-embedded sections were deparaffinised and then heated to 95°C for 10 min in 10 mFlow cytometry was also performed on cell suspensions prepared from tumour tissue and on the tumour cells grown in culture. The cells were incubated with the appropriate primary antibody and then with a secondary fluorochrome-conjugated antibody. Flow cytometry was performed using a FACScan Instrument and analysed with Lysys II software . Tumour cell suspensions were prepared from tumour tissue by grinding the excised tumour through a stainless-steel mesh  (−1) carmine red dye and 5% (w v−1) gelatin were injected into the tail veins of anaesthetised animals. The mice died within 30 s of injection and were then cooled below 4°C for 1 h in order to solidify the gelatin in the blood vessels. The tumours were excised and fixed in a solution of 10% formalin in saline. Sections (5 μm thick) from the fixed and paraffin-embedded tumours were then cut, deparaffinised and counterstained in 2% light green dye (Sigma).Vascular volumes were analysed using an axial strip sampling technique . Sectiong. The serum was removed and either assayed immediately for VEGF or stored at −85°C until assayed. Vascular endothelial growth factor was measured in tumour cells grown in culture by first harvesting cells from a growing culture, washing them in ice-cold PBS and then resuspending the cell pellet, containing approximately 2 × 108 cells, in 25 ml of fresh extraction buffer in a tight-fitting Potter homogeniser. The buffer contained 50 mM Tris-HCl, pH 8.2, 2 mM dithiothreitol, 2 mM EDTA and 1% Triton X-100. After 10 strokes with the pestle, the resulting cell extracts were kept on ice for 30 min and then centrifuged for 15 min at 2000 g, to remove any insoluble material. The supernatants were removed and either assayed immediately for VEGF or stored at −85°C until assay. Vascular endothelial growth factor levels in tumours were measured by first preparing tumour homogenates. After weighing, excised tumours were placed in 10 ml of ice-cold extraction buffer in a Potter homogeniser and homogenised using 25 strokes of the pestle. The resulting homogenates were kept on ice for 30 min and then centrifuged for 15 min at 2000 g. The supernatants were removed, passed through a 0.45 μm Millipore filter and either assayed immediately for VEGF or stored at −85°C until assay. Protein concentrations in the serum, cell and tumour extracts were determined by the method of Vascular endothelial growth factor (VEGF) was detected by ELISA, using an antibody raised against recombinant mouse VEGF and a kit purchased from R&D Systems, Inc., Minneapolis, USA. Information provided by the manufacturer indicates that this antibody can be used to determine the relative mass values for natural mouse VEGF. Serum was obtained from blood samples taken by intracardiac puncture. The blood was allowed to clot for 30 min before centrifugation for 10 min at 1000 1H-imaging) and 162 MHz (31P-spectroscopy), although only proton imaging was performed in this study. Animals were anaesthetised by intraperitoneal (i.p.) injection of Hypnorm/Hypnovel/dextrose–saline in the ratio 5 : 4 : 31 (10 ml kg−1 body weight) and immobilised in a cradle that held the tumour in the centre of the surface coil. Hypnorm was from Jansen Pharmaceuticals  and Hypnovel was from Roche . The dextrose–saline solution contained 4% dextrose and 0.18% saline. Dextrose–saline (0.5 ml) was also given i.p. to reduce animal dehydration. Anaesthesia was maintained, for up to 5 h, with 1.75-h i.p. injections of Hypnorm–dextrose–saline solution in the ratio 1 : 19 (5 ml kg−1 body weight). A flow of warm air was used to maintain body temperature. Tumour perfusion was measured by using a series of rapidly acquired T1-weighted spin echo images  to monitor the inflow and subsequent efflux from the tumour of the MRI contrast agent, gadolinium diethylenetriaminepentaacetate (Gd-DTPA) , as described previously , was administered intravenously (i.v.) through a tail vein catheter over a period of ∼30 s to give 200 μmol kg−1 body weight. Images were acquired immediately before and for up to 30 min after contrast agent injection from a 1 mm-thick slice and a field-of-view of 20 times; 20 mm2 over a 256 × 128 (phase encode) data matrix, that was zero-filled to 256 × 256 data points. For calculation of the paramagnetic contribution to the relaxation rate (R1p), an image with TR=3 s was acquired at 30 min after contrast agent injection. Image pixel intensities in the short TR experiments were converted to R1p values using the expressions derived by R1p is directly proportional to the concentration of the contrast agent and can thus be used to assess quantitatively the perfusion of a tumour by the contrast agent. These R1p maps were transformed into maps of Gd-DTPA concentration, using the relaxivity of Gd-DTPA at this field strength (3.88 mM−1 s−1)  superconducting magnet , interfaced with a Varian Associates UnityPlus spectrometer and Sun workstation running Vnmr 5.3B software. An unshielded gradient set (Varian Associates) was used with a home-built probe incorporating a two-turn surface coil probe (20 mm diameter) tunable to frequencies of 400 MHz  is conjugated to Gd-DTPA. The agent was prepared as described by b thymoma EL-4, which has been transfected with a vector expressing a full-length chicken ovalbumin cDNA  and 11.99±0.4  h, respectively. The small difference in these doubling times is not statistically significant. Following subcutaneous injection of 5 × 106 EL-4 or E.G7-OVA cells, C57BL/6 mice developed palpable tumours within 5 days  and of those E.G7.OVA tumours that arose from cells that had been implanted between nominal passage numbers 13–20 (n=28). However, E.G7-OVA tumours that arose from cells that had been implanted between nominal passage numbers 7–12 grew more slowly than EL-4 tumours and those E.G7-OVA tumours that arose from cells implanted between the later nominal passage numbers 13–20 . Twenty-three out of 73 (32%) E.G7-OVA tumours underwent spontaneous regression, including 18 out of 42 (43%) that developed from cells implanted between nominal passage numbers 7–12 and five out of 26 (19%) that developed from cells implanted between nominal passage numbers 13–20. This difference in the tumorigenicity of early and late passage cells was judged to be significant by a ψ2 test .The E.G7-OVA thymoma, a derivative of the H-2min cDNA , has beeP<0.01) higher levels of ovalbumin  when compared to a later passage  . The EL-4 cell line showed no significant detectable signal . The numbers represent fluorescence intensity in arbitrary units. Immunohistochemistry showed significant staining for ovalbumin in sections of E.G7-OVA tumours, but no detectable staining in sections of EL-4 tumours (data not shown). Flow cytometric analysis of ovalbumin expression on cells prepared from disaggregated tumours showed that ovalbumin expression was significantly higher in regressing E.G7-OVA tumours than in those that continued to grow  expressed significantly .Previous work has shown that CD8 tumours . CD68 isin vitro were measured by ELISA . Vascular endothelial growth factor was significantly higher (P<0.01) in the serum of tumour-bearing mice  than in controls (9.2±1.1 pg ml−1). There were two mice in each group and four measurements of VEGF per group. There were also significantly higher (P<0.01) levels of VEGF in E.G7-OVA tumour homogenates (16.9±0.4 pg mg−1 protein) than in EL-4 tumour homogenates (12.5±0.5 pg mg−1 protein) (two mice in each group and four measurements per group). The E.G7-OVA tumours arose from cells implanted at nominal passage 12 and were taken for extraction 13 days after tumour cell implantation. There was no significant difference in the levels of VEGF in extracts of E.G7-OVA and EL-4 cells, 4.8±0.6 and 5.3±0.2 pg μg−1 protein, respectively.The levels of VEGF in the serum of EL-4 and E.G7-OVA tumour-bearing mice, in EL-4 and E.G7-OVA tumour homogenates and in extracts of EL-4 and E.G7-OVA tumour cells grown In addition to the lymphocytic infiltrate observed in regressing E.G7-OVA tumours, there was also clear evidence of an angiofibroblastic response , with prFigure 2size see . The incT1-weighted, spin echo images, in which the increase in signal intensity was proportional to the concentration of the contrast agent. Typical image series for an EL-4 tumour and an E.G7-OVA tumour, which went on to regress, are shown in Figure 3vs time, for 5 EL-4, 5 E.G7-OVA progressive and 5 E.G7-OVA regressive tumours  than in either the EL-4 or progressive E.G7-OVA tumours. These data can be used to estimate perfusion quantitatively by converting the increase in image pixel intensities, in the presence of the agent, into R1p values, as described previously  increase in the perfusion of those E.G7-OVA tumours that went on to regress, when compared to EL-4 and progressive E.G7-OVA tumours. These data were also analysed to estimate tumour vascular volumes, using the model described by P<0.01) and progressive E,G7-OVA tumours . The vascular volumes of the progressive E.G7-OVA tumours were also significantly higher than those of EL-4 tumours (P<0.05). The vascular volumes determined by MRI were similar to those obtained using the carmine dye injection technique for the regressive EG7-OVA tumours, but significantly higher than those obtained using the dye in the EL-4 and progressive E.G7-OVA tumours  cells. Rejection was accompanied by significant infiltration of CD8+ T cells, which was approximately five times greater than in control tumours (The E.G7-OVA tumour model has been widely used in studies of tumour immunotherapy procedures (P<0.025) than tumours arising from later passage cells, which showed lower levels of ovalbumin   . What thT1-weigthed spin echo images (Measurements of tumour perfusion showed that this was significantly increased in those E.G7-OVA tumours that went onto regress when compared to the EL-4 parent tumour and progressive E.G7-OVA tumours. Tumour perfusion was assessed by monitoring the inflow of an intravenously injected MRI contrast agent, Gd-DTPA, using a series of rapidly acquired o images . The preThe vascular proliferation observed here in regressing E.G7-OVA tumours has been observed previously during the spontaneous regression of melanoma  and in oThe observation that tumour regression is preceded by an increase in vascular volume is at variance with an MRI study of allogeneic tumours in rat, where a decrease in tumour volume was shown to be preceded by a reduction in vascular volume (+ T-cell infiltration. Adoptive transfer experiments have shown that control of E.G7-OVA tumour growth depends on retention of CD8+ T cells within the tumour. Tumour growth continued when the cells migrated out of the tumour into the spleen and draining lymph nodes (Detection of earlier signs of impending regression might be possible by monitoring CD8ph nodes . MagnetiIn conclusion, we have demonstrated a noninvasive MRI method that, by detecting the vascular proliferation that precedes immune rejection, can be used to detect the early signs of this process. We propose that DCE-MRI could be used in the clinic to evaluate early tumour responses to immunotherapy. This will, however, require its evaluation in other preclinical models of tumour immune rejection in order to establish the generality of this vascular response. Consideration will also have to be given to selection of the most appropriate pharmacokinetic model used to derive the vascular parameters. An additional complication arises from the fact that tumour perfusion is often grossly heterogeneous and can vary considerably between different tumour types. Therefore, in the clinic DCE-MRI measurements would need to be carried out immediately before and in the days following administration of immunotherapy. While this might require several imaging sessions, this is nevertheless more practical than trying to measure reduction in tumour volume and, more importantly, may give an earlier indication of response.


Mononuclear leucocytes were separated by Hypaque--Ficoll from 60 unselected primary colorectal carcinomas, and then fractionated by rosetting with sheep erythrocytes, either alone (E) or coated with antibody and complement (EAC). The E-rosetting cells, putative T lymphocytes, were cytotoxic in vitro to autologous tumour cells in 18 of the 60 cases, whilst the EAC-rosetting cells were unreactive. This intrinsic T-lymphocyte anti-tumour immunoreactivity was significantly associated with the presence of "cuffs" of small dark lymphocytes at the mesocolic or pararectal edge of the primary tumours, but there was no correlation with antitumour cytotoxic lymphocytes in the patient's blood at the time of operation.


Attempts were made to induce tumour-specific immunity in syngeneic mice by treatment with x-irradiated  tumour or surgical excision of developing subcutaneous tumour grafts. Resistance was evoked against 6 tumours and this was relatively weak. With the remaining tumours, no resistance against the immunizing tumour could be demonstrated, even though the transplantation tests were made highly sensitive by the use of inocula of as few as 2 × 103 cells in pre-irradiated (400 rad) hosts. Sera from mice immunized against each of the tumours were tested against viable cells of the immunizing tumour by indirect immunofluorescence. In no instance did tumour antisera give a convincing reaction with tumour cells although alloantisera raised by hyperimmunization of H-2 identical and H-2 different donors with osteosarcomata consistently gave strongly positive reactions. The results are interpreted as illustrating the weak tumour specific antigenicity of radiation-induced murine osteosarcomata. The possibility that antigenic deficiency is a consequence of immunosurveillance in this tumour system is discussed.The immunogenicities of 15 murine osteosarcomata induced with a bone seeking radioisotope (


The vascularisation of human primary colorectal carcinomas was studied immunohistochemically using the endothelial cell markers CD31 and factor VIII-related antigen. Tumour sections were systematically scanned at a magnification of x 100 to find areas of intense neovascularisation. Microvessel counts within these vascular 'hotspots' were performed at magnification x 250. Regions in which tumour cords were surrounded by a collagen IV-positive basement membrane were compared with those in which this was absent and with normal mucosa. CD31 appeared to be a more sensitive marker for endothelial cells than factor VIII-related antigen (mean 185 +/- 59 and 120 +/- 38 microvessels mm-2). Within individual tumour sections microvessel counts in vascular hotspots with highest vessel density correlated significantly with microvessel counts in vascular hotspots with second highest vessel density (P < 0.01). Microvessel counts in tumour areas where collagen IV-positive basement membrane were absent exceeded those in areas where it was present (factor of 1.7) and those in normal mucosa (factor of 1.6). The differences in vessel density between individual tumours and the low variability in vessel density within individual tumours using this quantification technique allow us to investigate the prognostic value of vessel density in areas of intense neovascularisation in human primary colorectal carcinomas.


The effects of sodium pentobarbitone anaesthesia, the presence of a tumour, and local hyperthermia to a tumour-bearing leg, on the pharmacokinetics of MISO in the mouse are reported. Analysis of MISO and its metabolite Ro 05-9963 was by high-performance liquid chromatography. The plasma kinetics of MISO were largely unaffected by any of these treatments, but hyperthermia substantially reduced tumour concentrations of the drug. The effects of tumour site and size on unheated-tumour drug concentrations were also studied, and an increase in tumour size was shown to decrease tumour MISO levels, but to different degrees according to whether implanted in the leg or flank. Uniformity of MISO distribution throughout heated and unheated tumours was examined, and levels were found to be constant within tumours. The presence of a temperature detector in heated tumours did not affect their drug concentration.


The purpose of the present study was to explore the therapeutic potential of serial administration of shedding-inducing endotoxin in a mouse tumour bladder model. The studies were conducted with two variants derived from the MBT-2 tumour namely, T5 and T50, the latter being far more aggressive than the former. It was found that T5 tumours responded to intravesical lipopolysaccharides (LPS) instillation by a considerable reduction in their pace of growth (P < 0.0001) when treatment was initiated 3 days after tumour implantation, but not when started after 7 days. The T50 variant did not respond to LPS when treated 3 days after implantation, but a considerable reduction in rate of growth occurred when treatment was started after 1-2 days. Shedding induced by intravesically instilled LPS was found to retard considerably the progression rate of experimental bladder tumour.


Perfusion was performed by cannulation of the femoral artery and vein in rats bearing DS-sarcoma on the hind foot dorsum. Perfusion rate was adjusted to maintain a perfusion pressure of 100–140 mmHg throughout the experiment. Following equilibration, nifedipine was continuously infused for 30 min (8.3 μg min−1 kg−1 BW). During constant-pressure isolated limb perfusion, nifedipine can significantly increase perfusion rate (+100%) and RBC flux (+60%) through experimental leg tumours. ‘Steal phenomena’ in favour of the surrounding normal tissue and oedema formation were not observed. Despite the increased oxygen availability (+63%) seen upon application of this calcium channel blocker, nifedipine does not result in a substantial reduction of tumour hypoxia, most probably due to an increase in O2 uptake with rising O2 supply to the tumour-bearing hind limb. Nifedipine application during isolated limb perfusion can enhance tumour microcirculation and may therefore promote the delivery (pharmacokinetics) of anti-cancer drugs to the tumour and by this improve the efficacy of pressure-controlled isolated limb perfusion.Isolated limb perfusion allows the direct application of therapeutic agents to a tumour-bearing extremity. The present study investigated whether the dihydropyridine-type CaBritish Journal of Cancer (2002) 87, 1462–1469. doi:10.1038/sj.bjc.6600611www.bjcancer.com© 2002 Cancer Research UK Isolated limb perfusion (ILP) is a treatment modality for malignancies of the extremities in which the tumour-bearing limb is isolated from the patient's circulatory system and perfused separately. This procedure allows the administration of anti-cancer agents to the tumour at high doses with only a minimum risk of systemic toxicity. High-dose regional chemotherapy was one of the first concepts in ILP which was complemented more recently by the application of cytokines and immune-modulators frequently in combination with hyperthermia .The response to chemo- or cytokine-therapy may however be compromised by ‘biological’ factors such as tumour blood flow and microcirculation, tissue oxygenation or pH distribution. The individual tumour microenvironment is a paramount determinant in the outcome of non-surgical treatments . For exaOne group of drugs that might achieve these goals (and by this might increase the efficacy of ILP) are calcium channel blockers (CCB). In experimental tumour therapy, CCBs have been of broad interest because of their ability to modify tumour blood flow and improve oxygenation status.2 supply, CCBs also seem to be able to improve the O2 status directly by reducing oxygen consumption  have been shown to improve tumour blood flow, oxygen availability , 1989, tsumption .in vitro and in vivo by CCBs of the dihydropyridine type  into the dorsum of the hind foot. Tumours were used when they reached a volume of between 0.8 and 1.3 ml, 4 to 9 days after tumour implantation corresponding to a tumour mass of 0.5% of the body weight  housed in our animal care facility were used in this study. They received a standard diet and acidified water −1. This stock solution was added continuously at a rate of 0.5 ml h−1 kg−1 body weight  to the perfusion medium via a catheter placed in the femoral artery resulting in a nifedipine dose of 8.3 μg nifedipine min−1kg−1 body weight. An equivalent volume of the vehicle was infused into control animals.Under protection from direct light, 2 mg nifedipine  was dissolved in 10 ml 96% ethanol. Further dilution was made with isotonic saline resulting in a concentration of 0.1 mg mlsingle-pass ILP of the tumour-bearing extremity was performed  obtained from donor rats. Prior to use, blood was diluted to a haematocrit of 25% with bicarbonate-buffered oxypolygelatine , an isotonic colloidal blood plasma substitute solution which is used in the clinical setting. By using this drug for haemodilution an isotonic and isooncotic perfusate was obtained. Mean haemoglobin concentration (cHb) was 77 g l−1. Mean glucose and lactate levels were 6.1 and 6.9 mM, respectively. Blood was oxygenated with a humidified gas mixture containing 6% CO2, 36% O2 and 58% N2 (v v−1).A −1 i.p., Narcoren™, Merial, Hallbergmoos, Germany) and polyethylene catheters were surgically placed into the thoracic aorta via the left common carotid artery and into the right external jugular vein. Mean arterial blood pressure (MABP) was continually monitored through the connection of the arterial catheter to a Statham pressure transducer . Arterial blood gas analysis was performed before and during ILP experiments using a pH/blood gas analyser . Since the anaesthetic agent used is known to induce respiratory depression in high concentrations, the depth of anaesthesia was assessed by monitoring arterial blood pressure and blood gas status to ensure that these parameters remained within the physiological range throughout the experimental period.When tumours had reached the target volume, animals underwent general anaesthesia  was injected intravenously to prevent coagulation. An incision was made in one groin and the femoral artery and vein were exposed. After ligation of accessible collaterals, the femoral vessels were cannulated , flushed with 2 ml of warmed oxypolygelatine and then connected to the perfusion equipment. Blood flow in deep-seated collaterals was restricted by a groin tourniquet which was tightened upon commencement of the perfusion. Perfusate flow rate was varied (range: 0.27–2.63 ml min−1) to achieve a constant perfusion pressure (PP) of approximately 100 to 140 mmHg in order to maintain an adequate tissue perfusion which might otherwise be impaired with PP approximately 15 mmHg below the systemic MABP  was used to measure red blood cell flux (RBC flux). Details of this method have been described earlier by Although, attempts were made to maintain the PP at a constant level during ILP, slight pressure changes (±5–10 mmHg) occurred. In order to assess whether changes in RBC flux were due to variations in PP or the result of nifedipine-induced vasodilation, the relative tumour vascular resistance (TVR) was calculated as a measure of the resistance to flow. The TVR was defined by the ratio of the MABP (or PP) and the RBC flux. This parameter is suitable for assessment of changes in the vascular diameter from variations in tumour blood flow.2) was assessed polarographically using a flexible O2-sensitive catheter electrode  which was inserted into the centre of the tumour for continuous monitoring of tumour pO2. Before each experiment, the pO2 electrode was calibrated with room air in a chamber with constant temperature, taking the ambient barometric pressure into account.Mean tumour oxygen partial pressure , 15, and 30 min to assess pH/blood gas status  as well as glucose and lactate concentrations which were determined enzymatically using standard test kits .After the surgical procedure, animals were allowed to stabilise and measurements commenced once constant baseline readings for PP, RBC flux and tumour oxygen tension were obtained for at least 20 min . Thereafter baseline values for blood flow rate, PP, RBC flux and tumour pOM perchloric acid, centrifuged and the supernatant neutralised with 2 M potassium hydroxide and diluted 1 : 2 with the mobile HPLC-phase. Concentrations were then determined using reversed-phase high-performance liquid chromatography (HPLC) and UV-detection at 254 nm. The isocratic separation was performed by a Superspher RP 18 end-capped column  and a guard cartridge system (5×4 mm). The mobile phase consisted of 0.05 M ammonium dihydrogen phospate, 0.01 M tetrabutylammonium hydroxide and 11.5% acetonitrile (v v−1), adjusted to pH 6.4. The flow rate was 0.9 ml min−1 and the sample size 40 μl. Concentrations of all metabolites are expressed as μmol g−1 tissue wet weight. Wet weight was estimated from tissue samples as follows: tumours (perfused for 1 h) were excised from the hind foot dorsum, skin was removed and wet weight recorded. Tumours were dried at 60°C until constant weight readings were attained. The tissue water content was the same in perfused and in untreated tumours (82.1±0.2% vs 82.0±0.1%) of comparable volume. Oedema formation during ILP can thus be excluded.In an additional series of experiments, the tumour of the isolated perfused leg and of the contralateral hind limbs of the anaesthetised animals were surgically removed and rapidly frozen in liquid nitrogen immediately following termination of the perfusion procedure (30 min of equilibration plus 30 min of nifedipine or vehicle infusion) and the tumours subsequently removed. The tumours were ground to a fine powder and subsequently freeze-dried. Thereafter, glucose and lactate concentrations were assayed enzymatically using standard test kits . Concentrations of adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) were determined by high-performance liquid chromatography  to 81% of the baseline value. Thereafter however, the decrease was intentionally compensated by the increased perfusate flow rate resulting in a constant PP of between 130 and 135 mmHg. During nifedipine application, the perfusate flow rate had to be increased by almost 100% in order to maintain a constant PP  remained almost constant  were taken together, a linear correlation (r2=0.606) was seen between the O2 supply to the tumour-bearing leg and the O2 uptake into the tumour , and higher lactate levels in the isolated perfused limb tumours . However, the higher glycolytic rate during ILP did not have a strong impact on the bioenergetic status. In isolated perfused tumours (without nifedipine) the ATP levels were 0.66±0.08 compared to 1.18±0.13 μmol g−1 in the contralateral non-isolated-perfused tumours. Although the oxygen supply was restricted during ILP per se (due to the reduced haematocrit of the perfusate which results in a lower oxygen transport capacity), a stable bioenergetic status was maintained.As a result of the reduced haematocrit (25%) used in the perfusate, the oxygen supply during ILP −1 during nifedipine application vs 0.59±0.09 μmol g−1 in ILP tumours without nifedipine). However, since the oxygenation status was not improved by nifedipine neither the lactate levels (25.7±2.2 vs 27.0±3.5 μmol g−1) nor the ATP concentration (0.79±0.11 vs 0.66±0.08 μmol g−1) markedly changed during nifedipine treatment compared to tumours during ILP without nifedipine. Obviously, the increase in tumour blood flow had practically no impact on the metabolic or bioenergetic status of the tumour.With nifedipine infusion during ILP, tumour perfusion substantially increased and resulted in a considerably higher nutrient supply which was reflected by a higher  tumour glucose concentration  of chemotherapeutic agents can be expected. In addition, the deterioration and heterogeneity of tumour blood flow is responsible for hypoxia in tumours which in turn reduces the efficacy of oxygen-dependent chemotherapeutic agents. For this reason, a supportive treatment modality  which leads to a reduction of heterogeneity or an improvement of tumour blood flow and/or a reduction of hypoxia might be of clinical interest.−1 led to a two-fold increase in melphalan concentration in the tumour tissue. The effect of CCBs on tumour perfusion has been investigated extensively over the last two decades. 2 values measured in tumours of isolated perfused limbs were lower  than in non-perfused contralateral control tumours with comparable volumes  (2-supply conditions were restricted (compared to control conditions in non-perfused limbs). This is partially due to the lower cHb of the perfusate (74±2 g l−1) compared to whole blood. Control measurements in the normal subcutis of perfused hind limbs (non-tumour bearing) and non-artificially perfused legs also showed poorer oxygenation during ILP (median pO2=37 mmHg in subcutis during ILP and 49 mmHg in control legs). In many experimental ILP studies  . These d2-values of 30% would only be abolished if the flow rate were to be increased by a factor of 4 or more. An improvement in tumour oxygenation was reported in two studies by 2 by approximately 50% in the tumour center . 2 rose only slightly by 2–3 mmHg  the O2-uptake by the tumour increased linearly (2 consumption is dependent on the oxygen availability as long as a ‘saturation level’ of the O2 supply is not reached. An improvement in the convective O2 transport (by increased perfusion) will only reduce tumour hypoxia if the O2 supply greatly exceeds the consumption rate. Due to the reduced haemoglobin level during ILP, the oxygen transport capacity of the perfusate is diminished (0.11 ml O2 per ml blood during ILP vs 0.2 ml O2 per ml blood in controls) resulting in a restricted supply situation. Obviously, the O2 supply in the present study was markedly lower than the ‘saturation level’ and the convective O2 transport during ILP did not meet the demands of the tumour tissue. During ILP therefore, where the O2 transport capacity is reduced, an improvement in the perfusion rate of 50–100% is probably not sufficient to bring about a significant increase in the median tumour pO2.Since the oxygenation status of a tissue results from a dynamic steady-state between the oxygen supply and the cellular Olinearly . PreviouAs a result of the restricted oxygen supply during ILP (due to the reduced haematocrit of the perfusate), the lactate levels in tumours of isolated perfused limbs were significantly higher than in tumours of contralateral control limbs, indicating a much higher glycolytic rate. However, the higher glycolytic rate had only a minor impact on the bioenergetic status resulting in ATP levels which are comparable to those found previously in untreated tumours  and in tIn conclusion, nifedipine can significantly improve tumour perfusion during pressure-controlled ILP. ‘Steal phenomena’ in favour of the surrounding normal tissue and oedema formation were not observed. Nifedipine can enhance tumour microcirculation and may therefore promote the delivery (pharmacokinetics) of anti-cancer agents. Although the application of this calcium channel blocker increases oxygen availability to the tumour the improvement of perfusion by nifedipine does not result in a substantial reduction of tumour hypoxia. On the basis of these results, nifedipine application during ILP can be expected to increase the delivery of anti-cancer drugs to the tumour and by this improve the efficacy of pressure-controlled ILP.


Further groups of F1 hybrid with and without the tumour received spleen cells from non-tumour bearing parent line animals. The G.V.H. reactions induced in the four groups of F1 hybrid were compared and no significant differences were found. This was true in separate experiments, involving two mammary carcinomata and a 3-methylcholanthrene induced sarcoma, wherein the period of tumour growth in the parent line donor and F1 hybrid recipient was varied.In order to investigate whether the presence of a tumour was associated with immunodepression in the host, spleen cells from parent line animals with tumours were injected intravenously into F


P = 0.007). A similar tendency was found for the mean fractional allele loss . Of 20 tumours (65%) with LOH in several microdissected areas, 13 had identical losses at 1–4 loci within two or three areas, suggesting clonal development of these areas. Markers near RB, DCC, BBC1, TP53 and at D13S325 (13q21–22) showed higher loss rates in grades 2 and 3 (between 25% and 44.4%) compared with grade 1 (0–6.6%). Tumour-suppressor genes (TSGs) near these loci might, thus, be important for tumour progression. TP53 mutations were detected in 27%, but BBC1 mutations in only 7%, of samples with LOH. Evaluation of all 25 loci in every tumour made evident that each prostate cancer has its own pattern of allelic losses. © 1999 Cancer Research CampaignThe aim of the present study was to find out whether increasing malignancy of prostate carcinoma correlates with an overall increase of loss of heterozygosity (LOH), and whether LOH typing of microdissected tumour areas can help to distinguish between multifocal or clonal tumour development. In 47 carcinomas analysed at 25 chromosomal loci, the overall LOH rate was found to be significantly lower in grade 1 areas (2.2%) compared with grade 2 (9.4%) and grade 3 areas (8.3%,


Canine transmissible venereal sarcoma (CTVS) is a naturally occurring contagious neoplasm which can be transplanted with intact viable cells across major histocompatibility (MHC) barriers within the species and even to other members of the canine family, such as foxes, coyotes, and wolves. After 2 to 4 months of progressive growth the tumour regresses spontaneously in adults but metastasizes in immunosuppressed hosts and neonates. The mechanisms of how the tumour cells manage to overcome histocompatibility barriers so successfully for such a long period and yet succumb later are not known. In the present study we found that CTVS cells were not stimulatory to the lymphocytes of normal or tumour-bearing animals in mixed lymphocyte-tumour reaction (MLTR), although the lymphocytes from tumour-bearing animals in mixed lymphocyte-tumour reaction (MLTR), although the lymphocytes from tumour-bearing hosts responded well to either phytohaemagglutinin (PHA) or third-party allogeneic lymphocytes. Immunofluorescent antibody (IFA) assay of MHC antigens by monoclonal antibodies (MoAb) to monomorphic Class I and Class II MHC antigens showed that progressor tumour cells lacked the expression of the antigens whereas 30 to 40% of regressor tumour cells expressed them.


DMXAA , a novel anti-tumour agent currently undergoing clinical evaluation, appears to mediate its anti-tumour effects through immune modulation and the production of the cytokine tumour necrosis factor-alpha (TNF). Our previous studies have shown that thalidomide, a potent inhibitor of TNF biosynthesis that has numerous biological effects, including inhibition of tumour angiogenesis, unexpectedly augments the anti-tumour response in mice to DMXAA. We show here that thalidomide (100 mg kg(-1)) has no effect when administered with inactive doses of DMXAA, and that it must be given simultaneously with an active dose of DMXAA to have its maximum potentiating effect on the growth of the murine Colon 38 adenocarcinoma. To address the issue of whether inhibition of serum TNF production is important for potentiation of anti-tumour activity, we have tested three potent analogues of thalidomide. All three analogues, when co-administered with DMXAA to mice at doses lower than those used with thalidomide, inhibited TNF production and were effective in potentiating the anti-tumour activity of DMXAA against transplanted Colon 38 tumours. One of the analogues, N-phenethyltetrafluorophthalimide, was 1000-fold more potent than thalidomide and at a dose of 0.1 mg kg(-1) in combination with DMXAA (30 mg kg(-1)) cured 100% of mice, compared with 67% for the group treated with DMXAA alone. We also tested pentoxifylline and found it to suppress TNF production in response to DMXAA and to potentiate the anti-tumour effect of DMXAA. The results are compatible with the hypothesis that pharmacological reduction of serum TNF levels might benefit the anti-tumour effects of DMXAA and suggest new strategies for therapy using this agent.


Flow cytometric endothelial cells quantification was found to be more sensitive especially at lower levels of immunohistochemical microvessel density measurement. The current method was found to be applicable for various tumour types and has the major advantage that it provides a retrospective and quantitative approach to the angiogenic potential of tumours.Assessment of microvessel density by immunohistochemical staining is subject to a considerable inter-observer variation, and this has led to variability in correlation between microvessel density and clinical outcome in different studies. In order to improve the method of microvessel density measurement in tumour biopsies, we have developed a rapid, objective and quantitative method using flow cytometry on frozen tissues. Frozen tissue sections of archival tumour material were enzymatically digested. The single-cell suspension was stained for CD31 and CD34 for flow cytometry. The number of endothelial cells was quantified using light scatter- and fluorescence-characteristics. Tumour endothelial cells were detectable in a single cell suspension, and the percentage of endothelial cells detected in 32 colon carcinomas correlated highly  87, 344–347. doi:10.1038/sj.bjc.6600457www.bjcancer.comCancer Research UK© 2002  Angiogenesis is essential for the outgrowth of tumours and metastasis formation . It has It is likely that the difficulties with this method have lead to a substantial number of studies in which no correlation  in phosphate buffered saline (PBS) or 70% ethanol in water for 1 h at room temperature prior to digestion. After fixation, the sections were washed twice with 4 ml PBS and centrifuged at 400 g. The sections were digested by incubation in 1 ml of different concentrations  of collagenase  and dispase (Life Technologies) for time periods of 5–120 min at 37°C during repeated vigorous pipetting. After the digestion, the cells were washed in 10 ml PBS/0.5% bovine serum albumin (BSA), centrifuged at 400 g and further processed on ice. Cell preparations were microscopically evaluated for the condition of the single cell suspension. Propidium iodide staining  was used to control for intact cells. Cell pellets were incubated with 20 μl of appropriately diluted primary mouse monoclonal antibodies. EC were identified by EN4 anti-human CD31 antibody  and QBEND-10 anti-human CD-34 antibody . After two washings, the cells were incubated with 20 μl biotinylated rabbit anti-mouse Ig antibodies  for 1 h. Finally, the cells were stained with phycoerythrin (PE)-conjugated streptavidine  for 30 min. Analysis was performed on a FACS-Calibur . Both red (585 nm) and green (530 nm) fluorescence, forward light scatter and sidescatter were recorded simultaneously of 10 000 cells. Analysis of data was performed using CellQuest-software (Becton and Dickinson).Frozen tumour tissues were obtained from the stocks of the Department of Pathology. Of each tumour two 10, 30 and/or 50 μm thick 0.5–1 cm2O2 in methanol for 30 min. Sections were blocked for non-specific antibody binding with 5% BSA in PBS. The slides were incubated in above mentioned antibodies for 1 h. After incubation with avidine-biotin complex (Vector Laboratories Inc.) for 30 min, the slides were developed with diaminobenzidine (Sigma), counterstained with haematoxylin  and the slides were mounted in entellan (Merck). The microvessel density (MVD) was evaluated as described previously  for 1 h. Endogenous peroxidase was blocked by incubation in 0,3% H−1 collagenase and 5 mg ml−1 dispase  between the percentages of CD31+ and CD34+ cells was observed  from tumour biopsies .n=6). For MVD assessment by immunohistochemistry a variation of 26% of the mean was observed.The analysis of EC percentages in tumour tissues by flow cytometry was found to be highly reproducible. Variation in the percentage of EC obtained by flow cytometry was observed to be less than 5% of the mean, when six independent measurements were performed on one tumour. For example, one tumour analysed using CD31 antibodies revealed a mean percentage of CD31-positive EC of 12.3%  were analysed. Examples of low vascular density in a well differentiated colon carcinoma and high vascular density in a poorly differentiated colon carcinoma are shown in Figure 2r=0.74, P=0.001) was found when flow cytometric analysis was compared with MVD in vascular hotspots . The current method is not subject to these problems. It quantifies the number of EC in frozen tissues accurately and objectively. The high correlation with data obtained by immunohistochemical analysis of MVD proved the specificity of our method. Because the current method was set up to determine the number of EC in an entire tissue section, the data obtained should theoretically be most comparable to the data of the immunohistochemical detection of the total mean number of vessels . Indeed,It might be argued that the CD31 antibody also identified other cell types present in the tumour tissues such as leukocytes. However, the immunohistochemistry data did not show staining of leukocytes see . In addiWithin the group of colon carcinoma tissues, which was selected for representative cases of all Dukes stages, no correlations were found between percentage of EC and Dukes stage, tumour size or survival. This is most likely due to the study group size. Future studies in large numbers of tumour tissues will provide information on clinical parameters, which was beyond the scope of the present study. The current method is potentially also suitable for analysis of tumour EC phenotype. In future studies the number and phenotype of EC in tumour biopsies before and after chemotherapeutic and antiangiogenic treatment modalities will be subject to investigations. This may reveal fundamental insights into the regulation of angiogenesis or alternatively may provide surrogate endpoints of tumour treatments.


We investigated the potential antiangiogenic effects of the drug razoxane by measuring the changes in parameters estimated from H215O and C15O positron emission tomography (PET) to indicate alterations in vascular physiology. The study comprised 12 patients with primary or metastatic renal tumours >3 cm in diameter enrolled in a Phase II clinical trial of oral razoxane. Perfusion, fractional volume of distribution of water (VD) and blood volume (BV) were measured in tumour and normal tissue before and 4–8 weeks after treatment with 125 mg twice-daily razoxane. Renal tumour perfusion was variable but lower than normal tissue: mean 0.87 ml min−1 ml−1 (range 0.33–1.67) compared to renal parenchyma: mean 1.65 ml min−1 ml−1 (range 1.16–2.88). In eight patients, where parallel measurements were made during the same scan session, renal tumour perfusion was significantly lower than in normal kidney (P=0.0027). There was no statistically significant relationship between pretreatment perfusion and tumour size . In six patients scanned before and after razoxane administration, there was no statistically significant change in tumour perfusion: mean perfusion pretreatment was 0.81 ml min−1 ml−1 (range 0.46–1.26) and perfusion post-treatment was 0.72 ml min−1 ml−1 . Tumour VD and BV did not change significantly following treatment: mean pretreatment VD=0.66 (range 0.50–0.87), post-treatment VD=0.71 ; pretreatment BV=0.18 ml ml−1 (range 0.10–0.25), post-treatment BV=0.167 ml ml−1 . Tumour perfusion, VD and BV did not change significantly with tumour progression. This study has shown that H215O and C15O PET provide useful in vivo physiological measurements, that even highly angiogenic renal cancers have poor perfusion compared to surrounding normal tissue, and that PET can provide valuable information on the in vivo biology of angiogenesis in man and can assess the effects of antiangiogenic therapy.Measurement of tumour and normal tissue perfusion  Razoxane, also known as ICRF 159 ((±)-1,2-bis propane), is a chemotherapeutic agent, which inhibits cell division in the premitotic and early mitotic phases of the cell cycle . Hellman215O and C15O positron emission tomography (PET) to measure a range of vascular pharmacodynamic parameters.With the recent interest in chemotherapy agents with specific antiangiogenic effects, razoxane has been reinvestigated and has recently undergone a Phase II clinical trial in renal tumours . To provin vivo. Techniques have been developed using 15O labelled water and carbon monoxide to quantify regional perfusion, fractional volume of distribution of water (VD) and blood volume (BV) within tissues. The background to PET measurements of vascular parameters is reviewed elsewhere . Two measurements of perfusion and BV were recorded at each session with the patient in the same scanning position. Pretreatment measurements were compared with known tumour phenotype characteristics. Changes in perfusion, VD and BV after 4–8 weeks of treatment with razoxane were recorded and compared with clinical response criteria, which have been described in detail elsewhere  starting approximately 30 s before the infusion. During this time, continuous arterial blood sampling was acquired using an online BGO detector and automated pump at a rate of 5 ml min−1 to measure the arterial input function , and then transferred to a SUN SPARC Workstation for processing and analysis.Prior to scanning, patients were cannulated with an arterial line inserted into the radial artery. An i.v. line was inserted into an accessible vein in the other arm. An ECAT 931 08/12 PET scanner  was used, which measures 15 transaxial planes in 0.65 cm slices covering a 10.8 cm axial field of view. Patients were positioned according to the location of their tumour on a CT scan, and the position recorded to use for the second scan to ensure that the pre- and post-therapy regions of interest for analysis were the same. A 20-min transmission scan using 215O perfusion study, the dynamic images were summed over all time frames to generate signal-averaged ‘Add’ images, which enabled the anatomy to be visualised with improved signal-to-noise ratio. Since the C15O BV images are already summed static images, no further processing was required to improve visualisation. The blood flow Add images and BV images were compared with the pretreatment CT scan to aid the localisation of the tumour and other normal organs. Region of interests were then manually drawn on each transaxial plane of the PET blood flow Add images and/or BV images to identify the areas of tumour and normal tissue that were in the field of view. Many of the large renal cell masses had areas of necrosis seen as very low radiotracer accumulation. On the perfusion images, large central macroscopic areas of necrosis were avoided when drawing the ROIs, as these were considered to be nonviable tumour  software . For each H−1 ml−1 and VD for each tumour and normal tissue ROI sampled. The blood flow and BV scans were repeated in each session, and so the mean values were reported and used for all comparisons. The data for each type of ROI  were pooled and the pre- and post-treatment values were compared using a Student's paired t-test.For each ROI, the average measured radioactivity concentration of all pixels within the ROI was computed. In the case of the BV images, the mean concentration of each ROI was divided by the mean blood concentration obtained from discrete samples to determine the fractional BV within the ROI. In the case of the dynamic blood flow images, the averaged radioactivity in each ROI was calculated for each time frame to generate mean ROI time–activity curves. Nonlinear least-squares curve fitting of the ROI time–activity curves and the measured arterial input function was carried out using in-house software based on a modified version of the standard single-tissue compartmental model developed for the brain (15O scan (one patient) in the same session. One patient (patient 2) had abnormally high values for tumour perfusion both pre- and post-treatment. The PET scan of this patient demonstrated a very high radiotracer concentration in the tumour mass. The tumour was adjacent to the right renal hilum. It is probable that the ROI around this tumour mass was contaminated by surrounding vascular structure, such as the renal artery or vein. As the model used to calculate perfusion in this study is not accurate at high flow values, the data for this patient were excluded from the analysis.In all, 12 of the Phase II trial patients were enrolled for the PET study , and sev−1 ml−1 (range 0.33–1.67), mean VD of 0.74 (range 0.50–1.11) and mean BV of 0.18 (range 0.08–0.40). Of the six patients who had scans before and after treatment with razoxane (−1 ml−1 (range 0.46–1.26), while the mean post-treatment perfusion was 0.72 ml min−1 ml−1 (range 0.51–1.15). There was a reduction in perfusion following treatment in two out of six patients . The mean pretreatment BV was 0.18 ml ml−1 (range 0.10–0.25), while the mean post-treatment BV had virtually no change at 0.17 ml ml−1 . The mean pretreatment VD was 0.66 (range 0.50–0.87), while the mean post-treatment VD was 0.71 (range 0.63–0.82) with a slight increase that was not statistically significant (P=0.22).Pretreatment tumour vascular parameters varied between the 12 patients, with a mean perfusion of 0.87 ml minr=0.32, n=13) .In order to examine for any potential confounding influence of tumour size on vascular parameters, the relationship between perfusion and tumour size was assessed using the Spearman correlation coefficient. There was no statistically significant correlation between the pretreatment perfusion and size of a tumour as measured by a CT scan . In the four patients who had kidney perfusion measured both before and after treatment with razoxane, mean pretreatment values were 1.84 ml min−1 ml−1 (range: 1.18–2.88), while the mean post-treatment values were 1.35 ml min−1 ml−1 (range: 1.16–1.72). As for tumours, there was a reduction in post-treatment renal perfusion, but again this did not reach statistical significance (P=0.14). Pretreatment BV for renal cortex was similar to tumours with a mean value of 0.17 ml ml−1 (range 0.11–0.20) and a post-treatment mean BV of 0.16 ml ml−1 (range 0.09–0.19) with no significant change (P=0.14). Mean pretreatment VD was 0.69 (range 0.52–0.78), while there was no significant change in the post-treatment VD .Table 3−1 ml−1 (range 0.84–1.32), while the post-treatment perfusion was 1.28 ml min−1 ml−1 (range 0.94–1.62). Pretreatment BV was 0.63 ml ml−1 (range 0.51–0.76), while the post-treatment BV was 0.54 ml ml−1 (range 0.54–0.55).Only two patients had both pre- and post-treatment measurements made in the spleen. Hence, there were insufficient numbers to perform a statistical analysis. For these two subjects, the pretreatment perfusion was 1.14 ml min/antiangiogenic agent in a Phase II study. Results have recently been reported of a Phase I study of endostatin that incorporated PET measurements of tumour blood flow  in patients to assess the effect of a potential antivascularOne difficulty of assessing the effect of antiangiogenic agents is that standard measures of tumour response based on volume change may be inappropriate, as tumour volume is not expected to change with response. Our hypothesis, based on preclinical data, was that a course of razoxane would reduce perfusion, BV and VD. However, we found that in the patients studied, there was no statistically significant change in vascular parameters after 4–8 weeks of razoxane therapy. Although the lack of control data and small sample size limit the conclusions that can be drawn from this study, one interpretation is that a single course of razoxane has no effect on tumour or normal tissue vasculature in the inoperable renal cell carcinomas studied.Nevertheless, the observation of a reduction in perfusion in two out of six patients  is consivs normal kidney also contrasts with the observation that levels of the potent proangiogenic protein, vascular endothelial growth factor (VEGF), are 3–37 times greater in renal cancer compared to normal parenchyma (α (HIF-1α) (α stabilisation results from mutations in the von Hippel–Lindau (VHL) gene, which occur in the majority of clear cell renal cancers  . HypoxiaAnother observation from the study reported here is the lack of relationship between tumour size and pretreatment perfusion. Similarly, no statistically significant correlation was reported between blood flow and size for a series of advanced breast cancers  of the patients enrolled for the study. This is lower than the 21 out of 22 (95%) described for a single-centre Phase I trial (215O and C15O in human renal cell carcinoma. New drugs are starting to enter clinical trials that specifically target tumour vasculature, and different methods are required to assess their effects. This study demonstrates that PET techniques for measuring physiological vascular parameters are a potentially useful tool in this area providing quantitative data in tumours and normal tissue. The short half-life of 15O allows multiple measurements within the same scanning session, which enables multiple assessments and the study of fast-acting therapies. The study highlights aspects of vascular physiology of tumours, which are of potential importance in monitoring new therapies and understanding tumour angiogenesis.In summary, the work has shown the feasibility of measuring PET vascular parameters using H


In mice, i.v. C. parvum induces intravascular coagulation. This is a prolonged reaction lasting up to 7 days. It results in thrombosis in hepatic vessels with consequent hepatic necrosis, and thrombosis in pulmonary and splenic vessels. This may be important in the assessment of the tumour-inhibitory activity of C. parvum.


Attempts to improve the selectivity of anti-cancer agents by conjugating them to antibodies directed at tumour associated antigens have demonstrated tumour localisation but only limited therapeutic success. We report here the advantage of a 2-stage approach in which the first component combines the selective delivery of antibody with a capability to generate a cytotoxic agent from a second subsequently administered component. A bacterial enzyme, carboxypeptidase G2 (CPG2) was conjugated with F(ab')2 fragment of a monoclonal antibody directed at beta subunit of human chorionic gonadotrophin (beta-hCG) and injected into nude mice bearing hCG producing CC3 xenografts of human choriocarcinoma. Time was allowed for the conjugate to localise at tumour sites and clear from blood before injecting para-N-bis (2-chloroethyl) aminobenzoylglutamic acid. Cleavage of the glutamic acid moiety from this molecule by CPG2 released a benzoic acid mustard. Growth of the tumour which is resistant to conventional chemotherapy was markedly depressed by a single course of treatment. This demonstrates for the first time the potential of an antibody directed enzyme to activate an alkylating agent and to eradicate an established human cancer xenograft.


By immunohistochemical study, dThdPase expression on tumour cells was observed in all seven specimens with high dThdPase protein levels, but not in all 14 cases with low dThdPase protein levels (P < 0.05). In summary, these data indicated that dThdPase is up-regulated in advanced solid types of gastric cancer, suggesting that dThdPase production in carcinoma cells might be induced by the microenvironment. © 1999 Cancer Research CampaignPrevious studies demonstrated that the immunohistochemical expression of thymidine phosphorylase (dThdPase) was related with distant metastasis and disease progression. In this study we investigated the production of dThdPase/platelet-derived endothelial cell growth factor in gastric cancer quantitatively. In a total of 75 tumour tissues and 60 normal gastric mucosa specimens, dThdPase protein concentrations were determined by ELISA. The amount of dThdPase was significantly higher in the tumour tissue than in the normal tissue. Intratumoural dThdPase concentrations were significantly higher in Borrmann types I and II macroscopically, in poorly differentiated and solid type histologically, in the medullary type of the tumour stroma, and in the tumour-invading serosa. In the medullary type of the amount of tumour stroma, protein levels of dThdPase were positively correlated with the vertical diameter of the tumour (


New guidelines to evaluate the response to treatment in solid tumors using imaging techniques have major limitations and important implications for radiological workload. This consensus statement from the International Cancer Imaging Society (ICIS) reviews the RECIST criteria and addresses several challenges regarding tumour measurement. Recommendations are made regarding tumour measurement and other issues are raised. The growing need to introduce a multimodality approach to monitoring response is recognized. ICIS welcomes a dialogue with the authors of RECIST to address issues raised in this review. New guidelines to evaluate the response to treatment in solid tumours were published in the Journal of the National Cancer Institute in February 2000  was founded in 1999 in order to promote education in cancer imaging and its role in the multidisciplinary management of malignant disease. This rapidly growing society currently has over 200 members worldwide and holds an annual scientific meeting and postgraduate teaching course. The vast majority of the associate members are radiologists who have an interest in cancer radiology and the 38 full members are all specialist oncological radiologists who are international leaders in the field.We recognise three important purposes for evaluation of treatment response for reporting the outcome of clinical trials, that is, tumour response as a prospective end point in early clinical trials, tumour response as a prospective end point in more definitive clinical trials designed to provide an estimate of benefit for a specific cohort of patients and tumour response as a guide for the clinician and patient or study subject in decisions about continuation of current therapy. We also acknowledge the need to assess tumour response in daily clinical practice. Although the protocols for imaging to assess response for clinical trials are likely to differ considerably from those required to assess individual clinical response in routine practice, the key to accurate, reproducible assessment, for both types of practice, is the involvement of a radiologist experienced in oncological imaging. The assessment of response not only requires an approximate estimate of tumour size, or more precise measurement, but also requires an in-depth understanding of the complications of cancer therapies and a detailed knowledge of the disease-specific patterns of tumour recurrence. The difference between imaging in clinical care and imaging for clinical trials is subtle, especially for the non-radiologist, but of paramount importance to obtain results that are reproducible and consistent across centres. In some manner, the difference in radiologic assessment is similar to differences in practice for the chemotherapist practising in clinical care versus enrolling and caring for a patient on a clinical trial.best overall response but limited or no concordance between RECIST and WHO for time to progression  and the bidimensional method (WHO) is discrepant for progressive disease. The RECIST criterion of 20% increase in length to indicate progressive disease (PD) does not equate to the 25% increase as judged by the WHO criteria. Indeed a 20% increase in a unidimensional measurement using the RECIST criterion equates to a 44% increase in bidimensional measurement  state that measurement is to be made only in the axial plane for CT. With MR and multislice CT, longitudinal or oblique measurements as well as axial measurements can now be made readily. Three-dimensional (3D) assessment and measurement of solid tumours is now available in many institutions. Its widespread use will clearly depend upon penetration of the acquisition devices needed for these data as well as the advanced computer workstations needed to perform accurate 3D quantification. Three-dimensional measurement is not addressed within the new criteria but may be important in the future, particularly with the advent of highly sophisticated volumetric scanning techniques (including multichannel CT and volumetric MRI) capable of acquiring isotropic imaging data. The use of 3D volume measurement has already been substantiated in several studies  grow irregularly and also regress in an irregular manner. It is true, and has been well documented that at baseline or any one time point, there is good correlation between the maximal tumour diameter, bidimensional measurement and tumour volume. However, tumours do not necessarily grow or shrink in a rounded fashion. In fact, lymph nodes frequently grow to a greater extent in the short axis . While it is recognised that certain ‘cystic tumours’ increase in size on effective treatment . Interestingly, Cheson in a report of an international workshop to standardise response criteria for non-Hodgkin's lymphomas noted a paucity of data for the number of lesions to measure and included an arbitrary number in their criteria, but left open to further discussion. The critical issue is in fact the number of lesions that are needed to be measured to have a consistent response assessment—either by the same observer or another observer, that is, to decrease variability in response assessment based upon choosing a different set of lesions. Preliminary data and experience would suggest that for certain tumours it is not necessary to measure as many as 10 lesions . Reduction in the radiation dose could be achieved by limiting follow-up CT scans to the area(s) of disease, or the most likely sites of relapse  in order to modify and update the guidelines in accordance with current opinion as outlined above. We propose that such dialogue should be ongoing so that new information from existing and new technology can be introduced into the guidelines in the future in a timely and appropriate manner. For example, there is clearly a growing need to introduce a multimodality approach to monitoring tumour response, thereby integrating morphological and functional tumour measurements. The rapidly growing application of positron emission tomography (PET), PET/CT and MRI to monitoring tumour response provides this opportunity and opens the way to a new and exciting era in cancer imaging.


However, malignant cells which are not synthesizing DNA during exposure to the radiopharmaceutical will be spared. To determine whether tumour incorporation of [125I]IUdR could be enhanced by protracted administration, we used a C6 cell line, growing in the brains of Wistar rats, as a glioma model and compared three methods of intracerebral delivery of [125I]IUdR. Twenty-four hours after administration of drug, autoradiography of brain sections demonstrated nuclear uptake of the radiopharmaceutical in cells throughout tumour while normal brain cells remained free of radioactivity. The [125I]IUdR labelling indices (% ± s.e.m.) achieved were 6.2 (0.4) by single injection, 22.5 (4.1) using a sustained release polymer implant (poly(lactide-co-glycolide)) and 34.3 (2.0) by mini-osmotic pump. These results emphasize the need for a sustained delivery system as a prerequisite for effective treatment. These findings are also encouraging for the development of a sustained release system for radiolabelled IUdR for use in the treatment of intracranial tumours, particularly in the immediate postoperative setting. © 2000 Cancer Research CampaignThe Auger electron emitting agent 5-[


Changes in RBC flux with increasing MABP depended mainly on tumour size. In small tumours, RBC flux decreased with rising MABP whereas in larger tumours RBC flux increased parallel to the MABP. As a result of these volume-dependent effects on tumour blood flow, the impact of AT II on tumour p O 2 was also mainly tumour volume-related. In small tumours oxygenation decreased with increasing MABP during AT II infusion, whereas in large tumours a positive relationship between blood pressure and O 2 status was found. This disparate behaviour might be the result of the co-existence of two functionally distinct populations of tumour vessels. In small tumours, perfusion decreases presumably due to vasoconstriction of pre-existing host vessels feeding the tumour. In larger malignancies, newly formed tumour vessels predominate and seem not to have this vasoresponsive capability (lack of smooth muscle cells and/or AT receptors), resulting in an improvement of perfusion which is not tumour-related per se, but is due to the increased perfusion pressure. © 2000 Cancer Research CampaignPerfusion and oxygenation of experimental tumours were studied during angiotensin II (AT II) administration whereby the rate of the continuous AT II infusion was chosen to increase the mean arterial blood pressure (MABP) by 50–70 mmHg. In subcutaneous DS- sarcomas the red blood cell (RBC) flux was assessed using the laser Doppler technique and the mean tumour oxygen partial pressure (


Lipid peroxidation products can be cytotoxic. Our objectives were (1) to use two pro-oxidants (iron and a pro-oxidative drug) to selectively increase lipid peroxidation in the implanted human breast tumours of mice consuming fish oil and (2) to kill the cancer cells without harming normal host tissues. The theoretical basis for selective cytotoxicity is that normal cells are better able to handle oxidative stress than cancer cells. Male athymic nude mice, consuming an AIN-76 diet, were injected s.c. with MDA-MB 231 human breast carcinoma cells. Three weeks later, all mice had palpable tumours, 3-10 mm in diameter, and diets were changed to modified AIN-76 diets containing 19% menhaden fish oil and 1% corn oil with or without supplemental 0.3% ferric citrate. After 2 weeks, half of the mice on each diet  were injected (three times per week for 2 weeks) with the ether-lipid drug edelfosine (ET-18-OCH3). The concentration of lipid peroxidation products in tumours  was significantly increased by both ferric citrate and ET-18-OCH3. The TBARS in livers were not increased, nor was there evidence of other harmful side-effects to the host mice. The addition of iron enhanced tumour cell death whereas ET-18-OCH3 suppressed tumour cell mitosis. The use of iron supplementation combined with ET-18-OCH3 resulted in the slowest growth rate, lowest mitotic index, highest level of lipid peroxidation products and increased the cytotoxic index in tumours without detectable harm to the host. That iron supplementation increased tumour suppression beyond that expected from the increase in the concentration of TBARS in the tumour merits further investigation.


While each transfectant demonstrated substantially increased tumorigenicity and growth rate compared to vector controls, only VEGF121 produced a combination of significantly reduced total and perfused vessel spacing, as well as a corresponding reduction in overall tumour hypoxia. Such pathophysiological effects are of potential importance, since antiangiogenic agents designed to block VEGF isoforms could in turn result in the development of therapeutically unfavourable environments. If antiangiogenic agents are also combined with conventional therapies such as irradiation or chemotherapy, microregional deficiencies in oxygenation could play a key role in ultimate therapeutic success.Vascular endothelial growth factor (VEGF) is an intensively studied molecule that has significant potential, both in stimulating angiogenesis and as a target for antiangiogenic approaches. We utilised MCF-7 breast cancer cells transfected with either of two of the major VEGF isoforms, VEGF Since several promising antiangiogenic strategies rely on blocking either VEGF or its receptors . The volumes of the tumours were measured using calipers and the formula ½ab2 (where a and b are the major and minor tumour dimensions).MCF-7 cells were obtained from American Tissue Culture Collection , and MCF-7 cells that stably express VEGF or FGF-1 were generated by transfecting MCF-7 cells with VEGF7, was injected 1 min prior to freezing to preferentially stain cells adjacent to the vessels  were automatically acquired and digitally combined under three different staining conditions. First, images of the DiOC7 were obtained immediately after the frozen sections were sliced on the cryostat. Following staining, the section was returned to the same stage coordinates, and imaged for both hypoxia (EF5) and total vasculature . The total vasculature and perfused vasculature images were enhanced using colour segmentation to identify appropriate blood vessels , background-corrected, and image-analysed using Image-Pro software  .To determine whether expression of VEGF3), VEGF121 (440±30), VEGF165 (570±80), and FGF-1 (440±90). Compared to vector controls, total vessel spacing was significantly decreased in both the VEGF121 (P<0.001) and VEGF165 (P=0.001) tumours, but unchanged in the FGF-1 tumours  and FGF-1 (P<0.013) tumours, but not for the VEGF165 (P=0.15) (see μm) vs the periphery of the tumour cross-section. On average, neither total nor perfused vessel spacing varied significantly with depth into the tumour for any of the four tumour models (data not shown). However, two distinctly different vascular patterns were observed among VEGF165 tumours. In roughly half of these tumours, a central region of hypoxia developed that was surrounded by a densely vascularised peripheral rim of vessels  were also markedly different among the transfectants, as shown in 165 and FGF-1 tumous, but significantly reduced in the VEGF121 tumours (P=0.026) compared to vector controls. This decrease in hypoxia for the VEGF121 tumours is in agreement with the striking decrease in perfused vessel spacing observed for these tumours (121 tumours) decrease in perfused vessel spacing.Overall tumour hypoxia was characterised by measuring the mean intensity of the Cy3 conjugated antibody to the EF5 hypoxia marker. As summarised in  tumours , which cWhile VEGF is well accepted as an important modulator of tumour growth and vascular development, specific pathophysiological alterations associated with the different VEGF isoforms are less well understood. The current work reaffirms the notion that different VEGF isoforms lead to distinct differences in tumour vascular structure when compared at the same implantation site. In addition, we found that such vascular changes are, in some cases, directly associated with alterations in tumour oxygenation.121- and VEGF165-transfected glioma cell lines implanted either subcutaneously (s.c.) or intracranially (i.c.). VEGF165 transfectants grew much more rapidly than wild type at either location, with a corresponding increase in vascular density at both. Interestingly, VEGF121 transfectants exhibited enhanced vessel growth only when implanted orthotopically in the brain.In previous studies, results have varied widely in terms of both tumour growth rate and vascular density when different tumour models and implantation sites were considered. 121vs the VEGF165. In this model, VEGF120-overexpressing tumours tended to more effectively recruit systemic vessels, but failed to develop adequate internal vascularisation , VEGF165 tumours produced a much more densely vascularised plexus of blood vessels overall.Using transfected fibrosarcoma cell lines, 121 and VEGF165 transfectants were significantly higher than vector controls and essentially equal to each other, while FGF-1 tumours grew at a somewhat less rapid rate. Both VEGF121 and VEGF165 produced densely arcading networks of blood vessels of increased vascular diameter. In contrast to both the fibrosarcomas and melanomas, however, spatial heterogeneities in vascular spacing were generally not observed. On average, neither total nor perfused vascular spacing varied as a function of distance from the tumour surface for any of the MCF-7 transfectants, although roughly half of the VEGF165 tumours demonstrated a reduction in vasculature in the tumour centre compared to periphery. Also, in contrast to previous reports in other models, MCF-7 VEGF121 transfectants were much more evenly vascularised than the VEGF165, as measured by the reduction in vascular spacing. Although the reasons for these disparate findings are unclear, spatially dependent vascular heterogeneities could possibly be related to either specific implantation site or differences in tumour volume.In the current study, human MCF-7 cells were implanted orthotopically in the mammary fat pad. Growth rates of VEGF−1’ or ‘positive pixels mm−2', is that vascular spacing is more closely related to the ability of the blood vessels to uniformly supply the tumour with oxygen and nutrients. Especially in tumours containing an uneven distribution of vessels, determinations of mean vascular density can be highly misleading in terms of tumour oxygen delivery. For example, a tumour with a highly localised cluster of dense vascularisation could have an overall vascular density equal to that of a tumour having a reduced but homogeneous distribution of vessels. Clearly, microregional efficiencies in the delivery of either oxygen or chemotherapeutic agents would be quite different between the two. Such differences are apparent when using our ‘distance map’ measurements of vascular spacing, which depend on vessel number, size, and spatial distribution. Although neither perfused vessel spacing nor tumour hypoxia was significantly altered in the VEGF165 tumours, VEGF121 tumours demonstrated significant changes in both. This decrease in perfused vessel spacing suggests that these vessels are more efficiently distributed in the VEGF121 tumours, which is supported by the significant decrease in overall tumour hypoxia observed in these tumours.A key advantage in our method of measuring vascular spacing, rather than the more commonly reported ‘vessels fieldFinally, FGF-1 transfectants have also been reported to form large, vascularised tumours and to confer a more malignant phenotype upon MCF-7 cells, without oestrogen supplementation (121 and VEGF165 isoforms have such disparate effects on vascular structure and function among different tumour models. Although tumorigenicity and vascular growth were increased by both in all of the previously cited tumour models, specific alterations in vascular morphology were distinctly different. Interestingly, it has been reported that while VEGF121 is the predominant form expressed in human breast carcinomas (165 variant is predominant in glioblastomas (121 or VEGF165 transfectants of the three tumour types do not necessarily follow this same pattern. In breast tumours, the predominant variant, VEGF121, was also the more effective in inducing extensive tumour vascularisation when overexpressed in that model. In melanomas and gliomas, however, an entirely different relationship holds true, and in each case, the predominant isoform is the less important in terms of promoting vascular development (A major unanswered question raised by this and previous studies is why VEGF121 and VEGF165 may be related to variations in heparin binding, isoform size, or diffusivity (Previous studies have speculated that differences in vascular configuration between VEGF


In a retrospective study we have used an immunoperoxidase procedure to localize the glycoprotein fibronectin in human rectal carcinomas, concentrating on tumour invading thick-walled extramural veins. Fibronectin was present in 29 out of 38 cases, in connective tissue stroma, and was not in direct association with the tumour cells, except in areas of necrosis. We found no correlation between the presence or absence of stromal fibronectin and (1) the degree of cellular differentiation within the tumour, (2) tumour progression (Dukes' classification) (3) the subsequent development of metastases and (4) patient longevity. OUr results do not support the conclusions from in vitro studies  that the metastatic potential of carcinomas may be partly determined by the ability of tumour cells to synthesize pericellular fibronectin.


In patients with a variety of tumours, extensive washing of the lymphocytes from those cases with advanced disease was found to greatly enhance their specific cytotoxic effects. This specificity was restricted to autologous tumour cells and allogeneic cells of similar histological origin. This cross-reacting cytotoxicity was not, however, universal, especially in cases of malignant melanoma. The cytotoxicity evoked by washing was abolished by the addition of the patient's serum. This serum effect showed a similar specificity to that found for lymphocyte cytolysis. The effect of washing, and the specific inhibitory effect of serum, was not detectable in early cases of primary malignant melanoma. The serum component responsible for inhibiting lymphocyte cytotoxicity had no detectable affinity for the target cells and appears to act on the lymphocyte surface, implying that tumour antigen may well be implicated.In a microcytotoxicity assay the lymphocytes from cancer patients were tested on autologous and allogeneic tumour cells


I.p. administration at several dose levels over periods of up to 12 weeks, or continuous i.v. infusion of high doses of misonidazole (MISO) for 15 h, produced no significant change in peripheral nerve conduction velocity (NCV) and did not prevent the normal increase in NCV as the animals matured from 12 to 24 weeks of age. Peripheral NCV  was reduced in both MISO-treated and control mice with hind-limb tumour implants, presumably owing to physical pressure due to tumour growth. In addition, neither the medial nerves nor the tibial nerve in the normal limbs of the tumour-implanted, drug-treated animals showed any change. Consequently our earlier and present studies do not confirm the recent reports of changes in NCV following either acute or chronic MISO administration to mice.


We have previously reported that irradiation of the abdomen of mice before i.v. injection of both immunogenic and nonimmunogenic tumour cells is capable of suppressing their ability to form metastatic lung nodules in a time and dose-dependent fashion. Experiments with segmental exposure indicated the target organ to be located in the ventral half of the abdomen. The effect has now been shown positively to depend upon irradiation of the caecum, and can be abolished either by shielding the caecum from irradiation or by surgically removing it prior to irradiation. Further experiments have shown that the effect cannot be elicited in germ-free mice and that its magnitude is markedly reduced in animals given gut-sterilizing antibiotics. Split-dose irradiation only slightly reduced the magnitude of suppression, provided both doses were given within the time window of effectiveness of single doses. Tumour-growth retardation was observed and spontaneous lung metastases were also suppressed when tumour-bearing mice received abdominal irradiation 7 days after tumour cell transplantation into the leg. However, abdominal irradiation did not significantly reduce subsequent tumour transplantability by the s.c. or i.p. routes. The experimental data are consistent with a mechanism by which transmigration of enteric bacteria across the radiation-damaged mucous membrane of the caecum effectively results in an endogenous infusion of endotoxin.


O6-alkylguanine-DNA alkyltransferase (MGMT). There is considerable interest in improving the efficacy of such O6-alkylating chemotherapy by the prior inactivation of MGMT. We have examined the effect of the modified guanine base, O6-(4-bromothenyl)guanine  on MGMT activity and cell or xenograft tumour growth inhibition by temozolomide in the human breast carcinosarcoma cell line, MCF-7. PaTrin-2 effectively inactivated MGMT in MCF-7 cells (IC50 ∼6 nM) and in xenografts there was complete inactivation of MGMT within 2 h of dosing (20 mg kg−1 i.p.) and only slight recovery by 24 h. MGMT inactivation in a range of murine host tissues varied between complete and ∼60%, with extensive recovery by 24 h. PaTrin-2 (10 μM) substantially increased the growth inhibitory effects of temozolomide in MCF-7 cells (D60=10 μM with PaTrin-2 vs 400 μM without). In MCF-7 xenografts, neither temozolomide (100 mg kg−1 day−1 for 5 days) nor PaTrin-2 (20 mg kg−1 day−1 for 5 days) had any significant effect on tumour growth. In contrast, the PaTrin-2–temozolomide combination produced a substantial tumour growth delay: median tumour quintupling time was increase by 22 days (P<0.005) without any significant increase in toxicity as assessed from animal weight. A PaTrin-2–temozolomide combination may therefore be beneficial in the treatment of human breast cancers.Tumour resistance to chemotherapy involving methylating agents such as DTIC (dacarbazine) and temozolomide is linked to expression of the DNA repair protein  Standard first-line chemotherapy for advanced breast cancer results in disease regression in the majority of patients, but these responses are rarely complete or sustained  plays a key role in determining tumour resistance to these drugs ) and chloroethylating (e.g. bischloroethylnitrosourea (BCNU)) agents are well established in oncology and are a component of high-dose therapy regimens used in the treatment of breast cancer  in DNA, prior to the administration of a chloroethylating drug guanine  was prepared in dimethylsulphoxide  and diluted into culture medium just prior to use. For animal studies, PaTrin-2 was ground to a fine powder and suspended in corn oil at 4 mg ml−1 immediately prior to intra-peritoneal (i.p.) injection. Temozolomide was freshly prepared at 40 mg ml−1 in 20% DMSO/80% phosphate-buffered saline (PBS) and diluted in 0.9% NaCl solution.PaTrin-2 and temozolomide were provided by the Cancer Research Campaign Drug Formulation Unit, University of Strathclyde, Glasgow, UK. For cell culture studies, a stock solution of PaTrin-2 (20 m2/95% air.MCF-7 cells (a human breast adenocarcinoma cell line) were grown as a monolayer in RPMI medium containing 10% foetal bovine serum , at 37°C in a humidified atmosphere of 5% CO6) were incubated in the presence of increasing concentrations of PaTrin-2 at 37°C, 5% CO2. After 2 h, cells were pelleted and resuspended in 10 ml PBS. This was repeated three times in order to remove any residual PaTrin-2. Finally, cells were pelleted and assayed for MGMT activity as previously described  at 37°C, 5% CO2, increasing doses of temozolomide or vehicle were added and the cells were incubated for a further 4–5 days. At the end of the exposure period, 150 μg MTT was added to each well and plates were incubated for 3 h at 37°C, 5% CO2. The media were removed and the formazan crystals formed in the viable cells were solubilised in 200 μl DMSO. The absorbances at 540 and 690 nm were determined using a Titertek Multiscan ELISA plate reader and growth inhibition calculated as a percentage of the A540–A690 of untreated wells.To determine toxicity, the MTT  growth inhibition assay, based on the method of 3) were implanted in the right flank while the mice were under ethrane and halothane anaesthesia and the experiments begun when tumour volumes had reached a suitable size (see below). As MCF-7 tumours are oestrogen receptor positive, they required additional oestrogen for growth. To prepare oestrogen pellets, β-oestradiol (468 mg) was added to 9.7 g silastic and mixed. Curing agent (1.1 g) was added and the mixture spread into three (26 mm × 12 mm × 1 mm) glass formers. These were then incubated at 42°C overnight before being cut into 2 mm × 2 mm × 1 mm cubes (∼2 mg oestradiol per pellet). The pellets were stored at 4°C until insertion subcutaneously at the tail base, simultaneously with the tumour implant and monthly thereafter. Agents were injected i.p. within 15 min of preparation: Temozolomide was injected 1 h after PaTrin-2 or the vehicle control. Animals were cared for in accordance with Home Office guidelinesMale nude mice (O/Nu: outbred ALPK Nu/Nu) were purchased from Zeneca . Animals were housed in a sterile environment and allowed free access to food and water. MCF-7 human breast tumour xenograft samples  dissected out and immediately frozen in dry ice. Bone marrow was collected from femora, which were dissected from each mouse. A minimum amount of bone was trimmed from each end and a 21-gauge needle inserted through the epiphyseal cartilage and the bone marrow flushed into an appropriate volume of PBS. Tissue was stored at −70°C until assayed for MGMT activity.To measure MGMT depletion following PaTrin-2, six groups of mice with at least five mice in each group received PaTrin-2 20 mg kg−1 day−1); the PaTrin-2 only group were given PaTrin-2 (20 mg kg−1 day−1) then DMSO in PBS, and the PaTrin-2 plus temozolomide group were given PaTrin-2 (20 mg kg−1 day−1) then temozolomide (100 mg kg−1 day−1). Drugs or vehicles were administered i.p. once daily for 5 days with a separation of 1 h. Up to 10 and at least six animals were assigned to each group, and mean tumour volume was standardised across the groups at the start of the experiment: thus the control, PaTrin-2, temozolomide and PaTrin-2 plus temozolomide groups had mean tumour volumes of 29.8±7.6 (range 19.0–38.7), 33.2±14.7 (range 16.5–58.7), 35.1±10.9 (range 20.9–52.4) and 30.3±10.0 (range 20.7–44.5) mm3, respectively.To assess the ability of PaTrin-2 to sensitise human breast tumour xenografts to the tumour growth inhibitory effects of temozolomide, groups of at least six nude mice were treated as follows: the vehicle control group were given corn oil then 20% DMSO in PBS; the temozolomide only group were given corn oil then temozolomide  with measurements taken using digital calipers.The relative tumour volumes were plotted for each animal and tumour quintupling times (tqt) determined. The statistical significance of differences between treatment groups was evaluated using the Mann–Whitney test. The maximum relative weight losses observed in each group were compared using the Kruskal–Wallis test.−1 total protein). Exposure to PaTrin-2 for 2 h resulted in extensive inactivation of MGMT in MCF-7 cells: the concentration required to inactivate 50% of the MGMT was around 6 nM  nor PaTrin-2 alone had any significant effect on xenograft growth. However, the combination of PaTrin-2 and temozolomide resulted in a median tumour quintupling time of ∼43 days representing an increase of ∼22 days P<0.005; , Table 1O6-alkylating agent, temozolomide has been approved for the treatment of malignant glioma and is under consideration for use in melanoma, for which it is extensively prescribed, off license (e.g. The nse e.g.  and prosO6-alkylating agents are currently not as widely used in the management of breast cancer as they are in glioma, malignant melanoma and lymphoma, the possibility of overcoming alkylating agent resistance would make these agents useful additions to the chemotherapeutic management of this much more common malignancy. Attempts to improve the effectiveness of O6-alkylating agents therapy by prior inactivation of MGMT using methylating agents have foundered owing to the similar, and hence cumulative, toxicities of the inactivating and treatment drugs (While the O6-BeG and PaTrin-2, which very effectively inactivate MGMT in cells and tumours, improve the therapeutic effect of alkylating agents in a number of tumour models (O6-BeG in combination with BCNU have shown increased myelosuppression, requiring reduced doses for phase II studies (O6-BeG pretreatment of mice significantly increased the in vivo cytotoxicity and clastogenic effects of BCNU in bone marrow. It has also been shown that the granulocyte–macrophage precursor cells of primary human bone marrow samples are dramatically sensitised to the toxic effects of temozolomide by pre-exposure to O6-BeG (O6-BeG provides an increase in therapeutic index, that is, that the improvement in efficacy outweighs any additional toxicity (Strategies have therefore focussed on the use of nontoxic MGMT inactivators and it has been shown that in vitro than O6-BeG, and we considered it worthwhile to examine the extent to which PaTrin-2 could inactivate MGMT and increase sensitivity to temozolomide in a human breast tumour model as a prerequisite for any potential clinical trial in breast cancer. Our results show that PaTrin-2 is also a potent inactivator of MGMT in MCF-7 cells both in culture and in xenografts in vivo. Tumour MGMT depletion by PaTrin-2 was as extensive as was reported with O6-BeG in other tumour types (see PaTrin-2 is a more potent MGMT inactivator ypes see . We alsoThe MGMT inactivation by PaTrin-2 in MCF-7 cells resulted in marked sensitisation to temozolomide growth inhibition. Following implantation into immune deficient mice, the resulting xenografts are completely resistant to growth inhibition by a 5-day treatment regimen using temozolomide alone. This was probably a result of the resistance conferred by high levels of expression of MGMT. PaTrin-2 alone had, as anticipated, no effect on tumour growth rates. However, PaTrin-2 overcame the resistance to temozolomide producing highly significant tumour growth delays, but without increasing toxicity as judged by animal weights. Thus, the therapeutic index of temozolomide is increased by PaTrin-2 in this animal model. We have previously shown that human melanoma xenografts expressing moderate levels of MGMT do respond to growth inhibition by temozolomide, but this is considerably enhanced by pretreatment with PaTrin-2 . CurrentGiven the results of the xenograft studies with melanoma, and now breast cancer, it seems reasonable to speculate that the greatest benefit from PaTrin-2-mediated inactivation of MGMT might be seen in tumours with the highest levels of MGMT expression and inherent resistance to temozolomide. This might best be assessed in a phase II clinical trial in breast cancer, particularly since temozolomide alone has been shown to be ineffective in the MCIC trial. However, given the variable levels of expression of MGMT in this tumour type, it would be beneficial to assess MGMT levels in tumour biopsies from all patients in such a study so that the hypothesis that MGMT inactivation will be beneficial can be effectively tested.


Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme for degradation of 5-fluorouracil (5-FU). DPD activity is highly variable in liver and peripheral mononuclear cells (PMNCs) and it has not been well studied in human tumours. Characterization of DPD in colorectal cancer is of clinical interest through its role in the regulation of 5-FU, the main chemotherapeutic agent used in this disease. Therefore, DPD activity was analysed in colorectal tumour and adjacent normal tissue from 63 patients, including three liver metastasis. DPD activity was highly variable in all tissues studied (coefficient of variation 43-61%) and was higher in normal tissue than in tumour. The tumour-normal activity ratio ranged from 0.19 to 3.32 (median 0.76). PMNC DPD activity was available for 57 patients and was correlated with tumour activity (r(s) = 0.29, P < 0.001). A higher correlation was observed between PMNCs and tumour samples that were both obtained in the morning (r(s) = 0.49), consistent with circadian variation in DPD activity. Normal tissue DPD activity was not correlated with either tumour (r(s) = 0.11) or PMNC activity (r(s) = -0.06). This study provides the first analysis of DPD activity in colorectal cancer and illustrates the large degree of variation in tumour activity. The tumour-normal activity ratio results suggest that elevated tumour DPD can play a role in 5-FU resistance through increased inactivation in tumour cells, but is an uncommon event in colorectal tumours. The results support the use of PMNCs for monitoring tumour DPD activity, particularly when circadian variation is taken into account. As a large degree of the variation in tumour DPD activity is not explained by PMNC activity, more accurate alternatives are needed before DPD activity can be used for targeting 5-FU therapy.


As assessed by decrease in tumour volume and inhibition of tumour cell respiration and glycolysis, hyperthermia (intra-tumour temperature 42°C for one hour) potentiated the destructive effect of radiotherapy (1000 rad) on the allogeneic VX2 carcinoma in the hind limb of rabbits, and chemotherapy (methotrexate) produced a similar potentiation of irradiation. The resulting regression of the primary tumour in each case after dual therapy was comparable to that occurring after 3 applications of local hyperthermia, which has been shown to cure 50% of animals with this carcinoma. Combination therapy did not increase the survival time of the rabbits, however, all of which had lung and lymph node metastases at autopsy. The results focus attention on the relationship between a primary tumour and its metastases. The histological picture and the animal survival data suggest that the mechanism of tumour cell death and resorption of necrotic material following treatment may be important in enabling the host to deal with metastatic cells. After combination therapy, many metabolically and mitotically active cancer cells remained in the tumour mass, and the incomplete destruction of the primary tumour may have left the host with a burden of tumour cells too large to be destroyed by the immune system.