The number and size of tumours were compared with those in the control group. The change in tumour blood flow and the therapeutic effect of AC7700 on microtumours were observed directly by using Sato lung carcinoma implanted in a rat transparent chamber. AC7700 caused a marked decrease in the tumour blood flow of all LY80 tumours developing in various tissues and organs and growth of all tumours including lymph node metastases and microtumours was inhibited. In every tumour, tumour blood flow began to decrease immediately after AC7700 administration and reached a minimum at approximately 30 min after injection. In many tumour capillaries, blood flow completely stopped within 3 min after AC7700 administration. These results demonstrate that AC7700 is effective for tumours growing in various tissues and organs and for metastases. We conclude that tumour blood flow stanching induced by AC7700 may become an effective therapeutic strategy for all cancers, including refractory cancers because the therapeutic effect is independent of tumour site and specific type of cancer.In a previous study, we used subcutaneous LY80 tumours (a subline of Yoshida sarcoma), Sato lung carcinoma, and methylcholanthrene-induced primary tumours, to demonstrate that a novel water-soluble combretastatin A-4 derivative, AC7700, abruptly and irreversibly stopped tumour blood flow. As a result of this interrupted supply of nutrients, extensive necrosis was induced within the tumour. In the present study, we investigated whether AC7700 acts in the same way against solid tumours growing in the liver, stomach, kidney, muscle, and lymph nodes. Tumour blood flow and the change in tumour blood flow induced by AC7700 were measured by the hydrogen clearance method. In a model of cancer chemotherapy against metastases, LY80 cells  86, 1604–1614. DOI: 10.1038/sj/bjc/6600296www.bjcancer.comCancer Research UK© 2002  The ultimate goal of cancer chemotherapy is the attack and elimination of all cancer cells that develop in the body. The target of anticancer drugs has thus inevitably become the cancer cell. However, a chemical substance that shows strong cytotoxicity against cancer cells has similar effects against normal cells that are actively dividing, such as haemopoietic cells and alimentary canal mucosa cells, resulting in severe side effects. At present, very few drugs exhibit tumour-selective toxicity.Many researchers have considered the possibility that tumour growth might be suppressed by cutting off the supply of nutrients to tumours. Two approaches are currently being studied to accomplish this. One is an antiangiogenic approach, which was first proposed by The second approach was first suggested by Combretum caffrum and named it combretastatin A-4 (CS A-4). This compound was found to have potent inhibitory activity against tubulin polymerisation. Subsequently, the water-soluble prodrug combretastatin A-4 phosphate (CS A-4-P) was synthesised for in vivo testing. in vivo. Several recent studies reported that CS A-4-P showed antitumour effects against various kinds of tumours without severe side effects , 8–10 weeks old and with an average weight of 250–300 g, were used for TBF measurements and vital microscopic observations. Rats of the same strain, weighing 200–220 g each, were used for the therapeutic experiment. Rats were bred and maintained in a ventilated, temperature-controlled (24±1°C), specific pathogen-free environment, on a bed of wood shavings, with food and water freely available and a 12 h light–dark cycle. They were usually housed two or three per cage. The rats were fitted with transparent chambers for microscopic observations (see below) and were caged singly.Tumour cells that were used included LY80, a variant of the Yoshida sarcoma, and Sato lung carcinoma (SLC), an undifferentiated lung carcinoma. In our laboratory, LY80 and SLC are maintained by successive i.p. and s.c. transplantations, respectively. We chose SLC for vital microscopic observations because when the tumour is implanted in transparent chambers, demarcation between the edge of the growing tumour and normal tissue is clear, and therefore the change in microtumour size after therapy can easily be measured . For implantation of tumour cells in the liver or stomach, a midline incision 1.5–2 cm long was made. The tumour cell suspension was drawn into a 50-μl graduated syringe , and a 27-gauge syringe needle  was used to inject 10 μl directly into the left lobe of the liver or into the muscularis propria in the stomach. Injection depths were 2 and 1 mm, respectively, from the surface of the liver and stomach. The needle holes were sealed with synthetic resin glue . The incision wound was closed, and the animals were allowed to recover. For tumour cell implantation into the kidney, rats were anaesthesized and placed in the left lateral decubitus position, and a vertical incision was made in the right flank through the skin and peritoneum to expose the lateral aspect of the kidney. The tumour cells (2×106 cells in 10 μl) were implanted into the renal parenchyma by use of the above-mentioned syringe and needle. The site of injection was 2 mm below the renal capsule. After injection, the operation wound was closed in one layer with thread. For tumours growing in muscle, tumour cells (2×106 cells in 0.1 ml) were injected into the left biceps femoris as described for the kidney.LY80 cells growing in ascites of a donor rat were collected, suspended in phosphate-buffered saline, and adjusted to a concentration of 2×106 cells in 10 μl) was slowly injected into the middle of the ear by using a 27-gauge syringe needle. First, a tumour grew only at the site of the inoculation. When the largest diameter of the tumour was approximately 7 mm, primary tumours were removed by cutting off the ear. After a further 7–10 days, metastatic foci were formed in the anterior or posterior cervical lymph node.Lymph node metastasis was induced according to the method reported previously  was injected into the tail vein at a rate of 0.15 ml min−1 by using an infusion pump . Pentobarbital sodium  and enflurane  were used for anaesthesia. Pentobarbital was administered i.m. at a dose of 25 mg kg−1 10 min before the experiment, and supplemental doses (12.5 mg kg−1 i.m.) were given at 90-min intervals to maintain immobilization during the experiment. Enflurane concentration was maintained at 1% in the inhaled carrier gas at 1 l min−1 by means of an anaesthetic apparatus for small laboratory animals (AC7700 ((Z)-N-[2-methoxy-5-[2- vinyl] phenyl]-L-serinamide hydrochloride), one of the combretastatin derivatives, was synthesised and provided by Ajinomoto Pharmaceutical Research Laboratories, Kawasaki, Japan. The intensity of TBF stanching or antitumour activity was increased by replacing an OH group of the B ring of CS A-4 with an NHMABP was monitored in all rats in which TBF was measured. MABP was measured via a catheter  inserted into the right femoral artery. Pressure in the catheter was recorded with a pressure transducer , the output of which was fed into an amplifier  adapted for MABP measurement.−1), the blood flow value (in ml min−1 100 g−1 tissue) was calculated from the half-life of the clearance curve obtained. A tissue blood flow meter with two separate amplifiers  was used. Two hydrogen electrodes with 80-μm diameters  and two rod-type Ag/AgCl reference electrodes , which were inserted between the skin and musculature in the caudal region, were used for each rat. In some rats with tumours growing in the kidney or liver, one electrode was inserted in the tumour and the other electrode was inserted in the kidney cortex or liver within 3 mm of the tumour nodule, for simultaneous measurement of blood flow changes in both the tumour and the adjacent normal tissue.TBF was measured with the hydrogen clearance method  whose prBlood flow measurements in tumours implanted in the liver, stomach, kidney, or muscle were performed 7–10 days postinoculation. To measure the TBF of tumours growing in the liver or stomach, laparotomy was performed at the same abdominal wall as that used for the earlier incision, and electrodes were inserted into the tumour. Electrodes were inserted to depths of 3 and 2 mm from the surface of the liver tumour and the stomach tumour, respectively. For measurements of the TBF in the kidney tumour, a vertical incision was carefully made in the right flank through the skin and peritoneum, similar to the incision for implantation of the tumour, and electrodes were inserted into the solid tumour growing in the renal parenchyma. Electrodes were inserted to depths of 2 mm from the tumour surface. The TBF in the intramuscular tumours was measured by introducing electrodes to a depth of 3 mm below the tumour surface. For measurements of TBF in lymph node metastasis, a syringe needle  was used to make a pinhole in the skin overlying a tumour growing in the cervical lymph node, and a hydrogen electrode was inserted through the hole into the tumour tissue. The electrode was inserted to a depth of 3 mm from the surface of the skin. For TBF measurements of tumours in the kidney, muscle or lymph nodes, rats were placed prone on a heated stage at 34°C. For tumours in the liver or stomach, rats were placed in a supine position.Throughout the experiment, the animals were kept in the same position, and rectal temperature was monitored with a thermistor for small animals  and was maintained at 33.5–35.5°C. All rats were killed with ether at the end of the experiment. A check was performed to determine whether electrodes were located in the tumours.By means of the same method as described previously (Rat transparent chambers (−1) was administered via the lateral tail vein by using an infusion pump. The change in TBF and the tumour degradation process were directly observed via a light microscope  with a 10× ocular  and 2–20× objectives . Tumour vessels within the chamber were transluminated by a 12-V 100-W halogen lamp. The microscopic image was recorded using a closed-circuit video system consisting of a CCD video camera , a TV monitor , and a S-VHS video recorder . A video timer was superimposed on images for record keeping. Segments of the video tape containing desired images were transferred to computer hard disk . Final images were output by a digital printer .AC7700  were injected into 16 rats via the lateral tail vein at a rate of 0.15 ml min−1 using an infusion pump (Compact Syringe Pump). Tumour-bearing rats were divided into two groups: group I, to which the 0.9% NaCl solution was administered (eight rats); and group II, to which AC7700 at 10 mg kg−1 was administered (eight rats). Treatments were performed five times at intervals of 2 days, starting on day 7 after tumour cell injection. All rats were killed with ether and examined macroscopically 2 days after the last treatment. For microscopic examinations, brain, lung, heart, liver, spleen, kidney, gastrointestinal tract, and tumours were resected and fixed in 15% formalin, processed, and embedded in paraffin.As a model for systemic metastases, LY80 cells  and tumour regions were traced. The tumour area was calculated by using an area analyser . The weights of the skin tumours were measured with an electronic balance .The therapeutic effect of AC7700 on lymph node metastases was evaluated by use of the difference in the number and weight of lymph node metastases between the AC7700-treated group and the 0.9% NaCl-treated group. All tumours visible to the naked eye in the mediastinal, gastrointestinal, lumbar, adrenal, axillary, and inguinal lymph nodes were removed, and their weights were measured.This therapeutic effect was evaluated by assessing the difference in the growth curve between the AC7700-treated group and the 0.9% NaCl-treated group. Photographs of the tumour were taken before and 24 and 48 h after drug administration. A transparent vinyl sheet was placed over the photograph, and the tumour was traced onto the overlay. Tumour area was measured by an area analyser (WT-4400SE) and was expressed as a percentage compared with the area at 0 h.U-tests. P values of 0.05 or lower were considered significant.Results were expressed as means±s.d. The statistical significance of TBF reduction at each time point after AC7700 administration was evaluated with repeated measures ANOVA. Comparisons of tumour volume, weight, and number and of the body weights of the animals were made using Mann–Whitney −1 AC7700 or 0.9% NaCl solution are shown in Figure 1Tumour blood flow changes in tumours growing in the liver, stomach, kidney, and muscle and in lymph node metastases in response to i.v. administration of 10 mg kg−1 AC7700, it then began to increase and reached 157 mmHg. Meanwhile, TBF in the renal tumour decreased from 30.9 to 13.8 ml min−1 100 g−1 within 3 min. Blood flow in the renal cortex, however, was 157.5 and 150.7 ml min−1 100 g−1 before and after AC7700 administration, respectively. Thereafter, TBF continued to decrease, and it was approximately 8 ml min−1 100 g−1 after 6 h. However, renal cortex blood flow remained approximately 150 ml min−1 100 g−1 throughout the experiment.The TBF decrease caused by AC7700 in the renal tumour was completely tumour specific. Typical changes in clearance curves for the renal cortex and renal tumour after AC7700 administration are shown in Figure 2The effect of AC7700 on TBF in lymph node metastases was independent of tumour size, and the TBF of all tumours, including microfoci with a diameter of 4 mm or less, was completely stopped.In tumours within muscle, liver, and lymph nodes, no preexisting tissues remained within the regions measured. In particular, in lymph node metastases, even in very small growing foci (less than 4 mm in diameter), preexisting tissues were not observed (data not shown). In contrast, in renal tumours, even in tumours with a diameter greater than 8 mm, glomeruli survived within regions assayed   . No obvi−1 AC7700. Arterioles in normal tissue and arterioles feeding into tumours markedly contracted after AC7700 administration  in the control group. However, tumours completely stopped growing during 48 h of observation after administration of AC7700 (n=4). One example of the therapeutic effect of AC7700 against a small SLC tumour with a diameter of less than 3 mm is shown in Figure 8Figure 7f AC7700 –D. Histof AC7700 , F. In c−1 100 g−1. This is a noteworthy phenomenon that could be exploited as a therapeutic strategy against micrometastases in lymph nodes.The present study clearly demonstrated that i.v. administration of AC7700 strongly stanched TBF not only in subcutaneous tumours  but alsoSimultaneous measurement of blood flow in the renal cortex and the renal tumour showed that the TBF decreased markedly as a result of AC7700, but blood flow in the renal cortex hardly changed, even in regions only 3 mm from the tumour edge. However, TBF stanching in renal tumours was not as complete as that in tumours growing in other organs. The reason for this difference is probably related to the fact that many glomeruli, which naturally have high blood flow, sporadically survived in measured tumours.We previously showed (in vivo evidence for this suggestion has not yet been provided. Although it is not clear whether TBF stanching is caused by direct action of this compound on tumour vessels, it seems quite likely that some structural change would occur in tumour vessels in any site after AC7700 administration.It has not yet been sufficiently clarified why AC7700 causes TBF to stop and why the TBF stoppage is irreversible, whereas the decrease in blood flow in normal tissues is reversible. Some authors speculate that TBF stanching due to a combretastatin compound may be caused by a direct effect of the drug on tumour vessel endothelial cells (Tumour vessels, even large-calibre vessels, usually consist of only a monolayer of endothelial cells and have no pericytes or smooth muscle cells for a supporting structure; sometimes a basement membrane cannot be observed at all or only incompletely . The difIn summary, AC7700 stopped TBF at a dose that did not cause severe side effects regardless of the growing site of the tumour and markedly suppressed tumour growth. The efficacy of cancer chemotherapy based on TBF stanching is in theory independent of the type of cancer cell. Accordingly, this strategy may become a powerful therapeutic tool against refractory cancers, and AC7700 is a promising anticancer compound that shows such effects.


Administration of C. parvum alone did not improve the survival of C57BL/6 mice injected with the EL4 lymphoma. The anti-tumour effect of anti-EL4 Ig was however increased by C. parvum treatment, and the combination therapy of anti-EL4 Ig and cytotoxic drugs was even more improved. However, C. parvum only had this effect when given by the same i.p. route as the tumour cells, and the effect was greater when C. parvum was injected 5 days before than 1 day after tumour cells.


The vascular architecture of four different tumour cell lines  transplanted subcutaneously in mice was examined by means of microvascular corrosion casting in order to determine whether there is a characteristic vascular pattern for different tumour types and whether it differs significantly from two normal tissues, muscle and gut. Three-dimensional reconstructed scanning electron microscope images were used for quantitative measurements. Vessel diameters, intervessel and interbranch distances showed large differences between tumour types, whereas the branching angles were similar. In all tumours, the variability of the vessel diameters was significantly higher than in normal tissue. The quantitative data provide strong evidence for a characteristic vascular network determined by the tumour cells themselves. © 1999 Cancer Research Campaign


Considerable effort has been focused on devising methods for manipulating tumour oxygenation and thereby improving tumour radiosensitivity. The combination of nicotinamide and carbogen has been proposed to oxygenate both chronically and acutely hypoxic cells in tumours. However, results have varied markedly with both tumour model and measurement technique. The current objectives were (1) to determine whether changes in radiosensitivity following oxygen manipulation correlated with changes in tumour oxygenation and (2) to assess whether oxygenation was preferentially improved in specific tumour micro-regions. Using two murine tumour lines, the SCCVII carcinoma and the KHT sarcoma, tumour intravascular HbO2 saturations were measured cryospectrophotometrically following nicotinamide, carbogen or the combination. Generally, nicotinamide had minor effects on oxygenation, arguing against a substantial effect on acute hypoxia, while carbogen and the combination produced marked and equivalent improvements in oxygen availability. These results demonstrate that changes in tumour radiosensitivity may not agree with corresponding changes in oxygenation, even within a given tumour model, and that the efficacy of a given manipulative agent may vary substantially with tumour line. One possible explanation for these findings is that different subpopulations of clonogenic vs non-clonogenic cells may be oxygenated by alternative treatments.


Liposomal drug delivery enhances the tumour selective localisation and may improve the uptake compared to free drug. However, the drug distribution within the tumour tissue may still be heterogeneous. Degradation of the extracellular matrix is assumed to improve the uptake and penetration of drugs. The effect of the ECM-degrading enzyme hyaluronidase on interstitial fluid pressure and microvascular pressure were measured in human osteosarcoma xenografts by the wick-in-needle and micropipette technique, respectively. The tumour uptake and distribution of liposomal doxorubicin were studied on tumour sections by confocal laser scanning microscopy. The drugs were injected i.v. 1 h after the hyaluronidase pretreatment. Intratumoral injection of hyaluronidase reduced interstitial fluid pressure in a nonlinear dose-dependent manner. Maximum interstitial fluid pressure reduction of approximately 50% was found after injection of 1500 U hyaluronidase. Neither intratumoral nor i.v. injection of hyaluronidase induced any changes in the microvascular pressure. Thus, hyaluronidase induced a transcapillary pressure gradient, resulting in a four-fold increase in the tumour uptake and improving the distribution of the liposomal doxorubicin. Hyaluronidase reduces a major barrier for drug delivery by inducing a transcapillary pressure gradient, and administration of hyaluronidase adjuvant with liposomal doxorubicin may thus improve the therapeutic outcome. Liposomal doxorubicin may enhance the antitumour effect, and reduce the toxicity by improved pharmacokinetics compared with free doxorubicin. The polyethylene glycol (pegylated) coating reduces cellular reticuloendothelial system uptake and prolongs the circulation time .μl suspension of 2 × 106 human osteosarcoma cells from the cell line OHS  either orthotopically (o.t.) or subcutaneously (s.c.), by injecting 100 − 1 bodyweight  during all surgical and experimental procedures. Hyaluronidase  was injected intratumorally in various concentrations diluted in PBS to a final volume of 50 μl, or 50 μl (1500 U in 50 μl) was administrated into the tail vein. Liposomal doxorubicin  was injected into the tail vein 1 h after hyaluronidase administration, using 16 mg kg− 1 in 200 μl PBS.The mice were anaesthetised with an s.c. injection of Hypnorm/Dormicum/sterile water,1 : 1 : 2, 4 ml kg− 1). Pressures were monitored online using a MacLab analogue to digital recording system with a sampling rate of 4 Hz . Fluid communication between the needle and the tumour tissue was tested by compression and decompression of the polyethylene tubing and accepted when the IFP did not differ more than 20%.Interstitial fluid pressure was measured in orthotopic and subcutaneous tumours using the wick-in-needle technique  was injected intratumorally or i.v. The micropipette was withdrawn during the injection to avoid breakage of the pipette. Control tumours received 50 μl PBS alone, given intratumourally or i.v. Microvascular pressure was measured up to 100 min after the hyaluronidase injection, but was not measured continuously throughout the experiment due to replacement of the pipette because of frequent pipette breakage and clogging.The skin overlying the tumour was removed before micropuncture, and zero pressure was recorded in the saline film covering the wound. Microvascular pressure was measured in one vessel per tumour. After the first MVP measurement, 50 The MVP was only measured in s.c. tumours because at this tumour site it was possible to observe the microvessels after removing the overlying skin. The IFP was also measured in o.t. tumours as this is physiologically a more correct mode.2 and 5 μm thick tumour sections were mounted on glass slides.Mice were killed by cervical dislocation 2 days after injection of liposomal doxorubicin, the tumours were excised, embedded in tissue Tec , frozen in liquid Nμg ml− 1) for 1 h, followed by either biotinylated goat anti-rat IgG  (10 μg ml− 1) and streptavidin Alexa Fluor 633  (100 μg ml− 1), when colocalised with doxorubicin, or by rabbit anti-rat IgG (diluted 1 : 200) , goat anti-rabbit Dako Envision (100 μl)  and rabbit anti-goat IgG Alexa Fluor 488 (10 μg ml− 1) , when colocalised with hyaluronan.When staining blood vessels, the tumour sections were fixed in acetone for 10 min and incubated in goat serum to prevent nonspecific binding. The vessels were stained using rat anti-mouse CD31 antibody  (16.7 μg ml− 1), followed by incubation with stuptavidin Alexa Fluor 633 (10 μg ml− 1) for 30 min.When staining hyaluronan, the tumour sections were incubated with PBS containing 3% bovine serum albumin for 1 h to prevent nonspecific binding. Hyaluronan was labelled by incubating the sections for 1 h with biotinylated hyaluronan-binding protein  (10 μg ml− 1) for 5 min and rinsed in PBS for 1 min.When staining DNA, the tumour sections were equilibrated briefly in PBS and incubated with Hoechst 33258  . Colocalisation of doxorubicin and blood vessels was examined using an X20/0.3 objective. The colocalisation of doxorubicin and DNA was studied using an X63/1.2 water objective. The colocalisation of hyaluronan, DNA and blood vessels was studied using an X40/1.2 water objective. A 488 nm argon laser line was used to excite doxorubicin and rabbit anti-goat-IgG-Alexa488, a 633 nm HeNe laser line was used to excite streptavidin Alexa Fluor 633 and a two-photon mode locked Ti : sapphire laser  at Doxorubicin encapsulated in liposomes had a concentration that quenched the fluorescence. Doxorubicin does not bind covalently to the cells and was therefore washed away during the vessel labelling procedure. Colocalisation of doxorubicin and blood vessels had, therefore, to be assessed by sequentially imaging. The doxorubicin distribution was recorded before labelling the vessels, followed by imaging the same section and position with stained vessels. The sections were imaged along a radial track from the periphery to the centre. The start position of the track was marked, and based on this mark as well as the vessel structures the two images were easily overlayed. All images had a resolution of 512 × 512 pixels. Laser current and detector gain were chosen to minimise noise and to utilise all grey values.μm apart, and the first section was approximately 2 mm from the tumour periphery. Quantitative measurements require that the fluorescence is not attenuated nor bleached. As the sections were only 5 μm thick, attenuation will negligible. Bleaching of doxorubicin fluorescence was measured by scanning sections continuously for 2 min. No change in the fluorescence intensity was observed, demonstrating the absence of bleaching with the settings used.The uptake of doxorubicin was quantified by measuring the average fluorescence intensity per image. The fluorescence intensity was measured along a horizontal and vertical radial track from the periphery through the centre to the other periphery. In all, 6–16 images were recorded per section depending on the position of the section within the tumour and the size of the tumour. This was carried out for 10 sections per tumour and three tumours per group of treatment. Each section was 150–200 t tests .Curve fitting of theoretical models to experimental data was performed using Sigma Plot . The changes in pressures, relative to pretreatment values, were determined individually for each tumour, and then averaged. Gaussian pressure data were analysed using Student's P=0.05.Statistical analysis of doxorubicin fluorescence intensity data at each time point was performed using one-way analysis of variance, ANOVA. The frequency of images as a function of fluorescence intensity was analysed using the 5 × 2 contingency table and Chi-squared test. The contigency table contained a number of images with intensity values of 0–5 for the two groups, Caelyx™ alone or combined with hyaluronidase. All statistical analysis was performed using the significance criterion of n=6) (n=6), IFP was reduced to a lesser extent; approximately 20% (n=6) (P=0.052), the maximum reduction in IFP was significantly lower than the constant IFP level measured in control tumours injected with PBS (P<0.05). In addition, the reduction induced by 1500 U of hyaluronidase was significantly greater than the reduction induced by the other doses (P<0.05).Intratumoral injection of hyaluronidase in o.t. tumours reduced IFP in a dose-dependent manner up to a maximum reduction of approximately 40% after 1500 U of hyaluronidase (n=6) , 2. Howetely 20% . A furthτv) and a slow (τi) time constant  induced a slower reduction with time constants of approximately τv≈300 s and τi≈20 000 s.Maximum reduction in IFP was obtained approximately 1 h after hyaluronidase injection. The time response of the IFP reduction after intratumoral injection of hyaluronidase was biexponential with a fast  and for 1500 U , and almost significant for 3000 U . Interstitial fluid pressure was thus reduced to a greater extent in the smaller tumours.The maximum reduction in IFP correlated inversely with the tumour volume , except n=2) tumours induced approximately the same maximum reduction in IFP as the intratumoral injection of 1500 U hyaluronidase  as well as i.v. (n=7) injection of neither 1500 U hyaluronidase nor PBS (data not shown) had any significant effect on MVP in s.c. tumours .To obtain more quantitative data of doxorubicin distribution and uptake, the doxorubicin fluorescence intensity was quantified by estimating the average pixel intensity per image , and by The improved distribution of doxorubicin induced by hyaluronidase was demonstrated also by plotting the number of images as a function of fluorescence intensity. Tumours treated with liposomal doxorubicin and hyaluronidase had a higher number of images with detectable fluorescence than tumours treated with liposomal doxorubicin alone . A Chi-sDoxorubicin was located intracellularly, and hardly any fluorescence was seen in the ECM. The intracellular distribution of doxorubicin was mainly located in the nuclei, shown by colocalisation of doxorubicin and DNA . HyaluroDoxorubicin is one of the most effective drugs in the management of osteosarcoma . Althougτv) reflects increased transcapillary transport, whereas the slower time constant (τi) reflects interstitial transport . In addition to high IFP, the poor distribution may be due to heterogeneous vascularisation and perfusion, slow interstitial diffusion and efficient extravascular binding of doxorubicin mainly in the tumour and endothelial cells.The distribution of liposomal doxorubicin in untreated tumours was heterogeneous, and mainly located in the periphery of the tumours. This correlates spatially with the peripheral IFP gradient previously measured in subcutaneous OHS xenografts  increased the uptake of Caelyx™ in the central part of the tumour two to four times (The present work demonstrates that hyaluronidase reduces the barrier for drug delivery by inducing a transcapillary pressure gradient. An efficient transcapillary pressure gradient is especially important for the extravasation of large molecules such as liposomes. Pre- or coadministration of hyaluronidase adjuvant with liposomal doxorubicin may thus improve the therapeutic outcome.


A method has been developed for determining the mean volume of breast cancer in women at the time of the involvement of the first, second, third,... nth axillary lymph nodes. It has been found that the proportion of patients with axillary involvement as well as the number of involved nodes increase progressively with tumour size. This orderly involvement of axillary nodes is observed in all patient subsets despite a wide spread of tumour volume at the time of invasion of the axillary nodes. This makes it possible to compute for each patient or subset of patients the size of the tumour at the time of the first node involvement, a parameter which characterises the propensity for nodal involvement. A strong correlation was demonstrated between the propensity to lymphatic involvement and the probability of distant dissemination. During tumour progression the capacity for lymphatic spread is on average acquired much earlier than the capacity for haematogenous spread. For tumours of the outer quadrants, the volume at first axillary involvement is smaller than for tumours located in the inner quadrants, whereas the tumour volumes at the time of distant metastatic initiation are equal for the two tumour sites. The discrepancy between these two observations shows that axillary involvement, while being a good index of the propensity of the tumour cells to acquire the capacity for distant spread, is not the cause of this spread. From a clinical point of view, these data show that the prognostic significance of axillary involvement can be further increased by taking into account the size of the tumour. The data suggest that there is a continuum from slow growing disease with late axillary involvement and late distant dissemination to the most aggressive subtype.


Intravascular leiomyosarcoma is a rare tumour entity originating from venous vessel structures and most frequently affecting the inferior vena cava.A 69-year old patient presented with a biopsy proven leiomyosarcoma of the right supraclavicular region. Tumour resection and histological assessment verified the intravascular tumour origin arising from the internal jugular vein and extending into the surrounding soft tissue.In the presence of a biopsy proven diagnosis of leiomyosarcoma the rare condition of an intravascular tumour origin has to be considered even without signs of venous stases. This may result in an altered surgical strategy. Microthrombembolism and pulmonary metastases may complicate the course of the disease. In contrast to liposarcoma and NOS sarcoma (pleomorph sarcoma not otherwise specified) previously known as malignant fibrous histiocytoma (MFH leiomyosarcoma) leiomyosarcoma only account for a small proportion of malignant soft tissue tumours in adults. References in the current literature vary between 5–10% [Four main locations for tumour origin of leiomyosarcoma can be distinguished: 1. Intraabdominal/retroperitoneal 2. cutaneous 3. subcutaneous and 4. vascular. The very rare intravascular growth pattern most frequently affects the retroperitoneum especially the vena cava inferior [Clinical symptoms derive from tumour growth with palpable masses or intraluminal obstruction leading to signs of venous stases and thrombosis. Extracaval venous branches are rarely the primary source of vascular leiomyosarcoma and involve venous branches of the lower extremity .In this report, we describe a case of a 69-year old patient with a primary intravascular leiomyosarcoma of the internal jugular and subclavian veins. Differential diagnosis, clinical and pathological criteria for diagnosis of these rare intravascular tumours will be discussed.A 69-year old female patient, with a previous history of hypertension, thyroidectomy due to hyperthyroidism and hysterectomy for uterus myomas, presented with a progressive swelling of the dorsal aspect of the right side of her neck without signs of vascular obstruction or venous stases. No abnormalities of neural status of the head and neck were observed. There was no functional or sensory loss of the right upper extremity. No signs of Horner's syndrome, dysphagia, cough or dyspnoe were evident. CT scan demonstrated a retroclavicular soft tissue tumour with a cranio-caudal extension of up to 4.5 cm which partially displaced the trachea to the left and compressed the subclavian vein. An adjacent tumour of dimensions 3.5 × 3.5 cm not clearly separated from the before mentioned tumour was located at the inferior right thyroid lobe, compressing the internal jugular vein. Near the confluence of these vessels a subtotal occlusion of the brachiocephalic vein is revealed Fig. . The MRISurgical exposure was obtained via a triangular incision running from behind the right ear, along the anterior axillary line and across the sternum. First, the brachial plexus was dissected, the phrenic and recurrent nerves identified and followed distally. The upper border of the tumour became visible at the upper thoracic aperture. The recurrent nerve was observed to run through the tumour capsule. Further preparation was carried out from the distal edge of the wound. The pectoralis major muscle was elevated and care was taken to preserve the vascular pedicle . It was further observed that the first intercostal space was invaded by the tumour. Subsequently a thoracic wall resection including a partial resection of the right clavicle, the right half of the sternum and the costal attachment of the first three ribs was performed uncovering the mediastinum. The vena cava was revealed and trachea dissected. In this area the tumour was in close proximity to the trachea displacing it to the left but without tracheal infiltration. Next, the carotic artery and the jugular vein were exposed.The tumour, located in the right supraclavicular region/upper mediastinum, was found to surround both the subclavian and the internal and external jugular vein. Hence a resection of the subclavian vein proximal to its conjunction with the superior vena cava was required. The internal as well as the external jugular vein were incorporated into the tumour conglomerate Fig. . The tumWithin the surgical specimen multiple nodular polypoid tumour masses of soft consistence with diameters of up to 3.6 cm, immediately adjacent to vascular structures of the subclavian, internal jugular and brachiocephalic vein were present. The tumour with its intravascular and extravascular components comprised a total area of 7.6 × 8 × 3.3 cm. The largest intravascular tumour sprout extended close to the resection surface of the vessel.The macroscopic appearance resembled an intravascular tumour originating from the subclavian vein with infiltration of extravascular structures.Microscopically the spindle-shaped cells of this mesenchymal neoplasm originated from the media of the venous vessel wall Fig. . The tumThe neoplasm derived from the media of the vessel wall, disrupted the existing vascular architecture and formed an intravascular tumour sprout.Immunohistochemically the majority of tumour cells were positive for smooth muscle actin and desmin. A positive reaction for the proliferation marker Ki 67 was found in 25% of all tumour cells,Thus confirming the diagnosis of an intravascular leiomyosarcoma Postoperatively only mild signs of mixed venous and lymphatic stases of the upper extremity following the resection of the subclavian vein were observed due to the well established collateral blood flow (as seen in the preoperative angiogram). These symptoms could be positively influenced by elastic compression dressings and physical lymph drainage. Owing to the resection of the right recurrent nerve, right sided vocal cord palsy occurred. Logopaedic training was initiated. The patient recovered well and was discharged two weeks later. Both pre- and post-operatively no symptoms of pulmonary embolism were detected.Unfortunately the patient declined the recommended radiation therapy.After an initial 5 month of tumour free survival without evident signs of either local or systemic metastasis a tumour relapse was detected. At this stage the patient refused further treatment apart from a palliative chemotherapy.Vascular leiomyosarcoma represent only a small proportion of soft tissue leiomyosarcoma . These rLeiomyosarcoma deriving form smaller vessels are an exception which may lead to nervous or arterial compression due to increased pressure within the neurovascular sheets. These tIn the patient collective of the plastic surgery department at the University of Bochum out of the 90 soft tissue leiomyosarcoma 8 cases presented with a clear vascular origin of the tumour. In the above described case, the tumour was localized in the internal jugular and subclavian vein, in the remaining 8 cases the tumours were found in the femoral vein.In the current literature unusual manifestations of intravascular leiomyosarcoma were described for venous branches of the lower extremity  whereas A study of 42 patients with leiomyosarcoma of the deep somatic soft tissue indicates that the predominant source of these rare malignant tumours are the small venous structures .The clinical picture of an upper venous stasis may be caused by a number of different malignancies such as lung cancer and lymphomas . In partMoreover CT scan and MRI will not in all cases allow for an exact image of the endovascular tumour component  as in thSuch tumour localizations result in both pre- and postoperative pulmonary microthrombembolism as frequent complications particularly in tumours of the pulmonary artery .Furthermore, pulmonary metastases as the preferred distant tumour manifestation must be considered in the oncological care and staging .As a malignant mesenchymal tumour, leiomyosarcoma displays differentiation tendencies towards smooth muscle morphology. Hence, histologically spindle shaped cells with eosinophilic cytoplasm with muscular striation and cigar shaped rounded nuclei can be observed. The cytoplasm is rich in contractile fibers (proteins) such as actin, desmin as well as h-caldesmon.The differential diagnosis includes the spectrum of spindle cell shaped neoplasm. Mesenchymal tumours, the benign and malignant tumours of the nerve sheaths myofibrolastic tumours , synovial sarcoma, fibrosarcoma and NOS (not otherwise specified) sarcoma have to be considered .In addition to histomorphology using standard H&E staining immunohistochemical staining for smooth muscle markers facilitates the correct diagnosis. The intravascular leiomyomatosis is characterized by the proliferation of smooth muscle vascular structures of the uterus or its surrounding. Intimal sarcoma, malignant mesenchymal tumours of the large arteries which originate from the intimal layer of the vessel wall and present as fibroblastic or undifferentiated sarcoma  and the Complete surgical resection of the vessel segment is the therapy of choice. When an intravascular tumour origin is suspected, a ligation of the vessel far distant from the palpable tumour mass might be necessary due to considerable expansion of the intraluminal tumour sprouts ,10.Leiomyosarcomas of a vascular origin appear to be associated with a more aggressive tumour growth and poorer prognosis compared to respective tumours of the soft tissue . IncomplLeiomyosarcomas of the inferior vena cava appear to have no adverse prognosis compared to other tumour localizations .The intravascular growth of the sarcoma predisposes for hematogenic metastases . Hence pIn this report, we have presented a rare case of intravascular leiomyosarcoma in the uncommon anatomical site of the upper extremity. Such diagnosis requires a complete tumour resection as the main treatment strategy, however such approach may not be fully effective due to difficulties associated with achieving clear resection margins. The intraluminal expansion of the tumour sprout may be considerable requiring vascular grafting to bridge longer vessel segments.The occurrence of malignant intravascular tumours may present as venous obstruction and mimic the symptoms of venous thrombosis. However, in the absence of venous stases in the rare instance of a leiomyosarcoma and close proximity to vessel structures, a rare event of an intravascular tumour origin must be considered.Written informed consent was obtained from the patient for publication of this case report and any accompanying images.The authors declare that they have no competing interests.DT conceptualized the case report, gathered the data and wrote the manuscript. ML drafted and revised the manuscript. JH gathered the clinical data and assisted with postoperative care of the patient. AD reviewed the literature. DH performed the initial surgery and took responsibility for the patient's care. MT assessed the histological specimens. HS conceptualized and supervised the process of data gathering and revised the final. CK assessed the histological specimens, aided drafting and manuscript revision. All authors read and approved the final manuscript.


In the process of tumour progression genetic instability is the basis for the evolution of tumour cell clones with various genotypic and phenotypic characteristics causing heterogeneity. Renal cell carcinoma has a long prediagnostic growth period, which increases the probability of clonal evolution. We have studied 200 consecutive renal cell carcinomas, addressing the interrelationship between intratumour heterogeneity and clinicopathological factors. DNA ploidy patterns were analysed in multiple samples from each tumour using flow cytometry and compared with clinical stage, tumour invasion, metastatic rate and survival. Eighty-five of 192 evaluable tumours (44%) were homogeneous concerning DNA ploidy . Among 107 heterogeneous tumours a majority (79%) contained aneuploid as well as diploid cell clones. Homogeneously diploid tumours had a lower incidence of local tumour spread compared with tumours with aneuploid cell clones (P < or = 0.001), but the frequency of distant metastasis at time of diagnosis was similar. The presence of aneuploidy in at least one sample from a tumour was a significant adverse prognostic factor (P < 0.001), whereas the degree of heterogeneity had no influence on survival. The frequent heterogeneity demonstrated indicates that multiple samples must be investigated to evaluate properly the malignant character of renal cell carcinoma.


We have examined apoptosis and proliferation in lymph node cell suspensions from patients with B-cell non-Hodgkin's lymphoma using flow cytometry. A method was developed which allowed estimation of the fractions of apoptotic cells and cells in the S-phase of the cell cycle simultaneously with tumour-characteristic light chain expression. Analysis of the tumour S-phase fraction and the tumour apoptotic fraction in lymph node cell suspensions from 95 B-cell non-Hodgkin's lymphoma (NHL) patients revealed a non-normal distribution for both parameters. The median fraction of apoptotic tumour cells was 1.1% . In the same samples, the median fraction of apoptotic normal cells was higher than for the tumour cells . The median fraction of tumour cells in S-phase was 1.4% , the median fraction of normal cells in S-phase was significantly lower than for the tumour cells . When the number of cases was plotted against the logarithm of the S-phase fraction of the tumour cells, a distribution with two Gaussian peaks was needed to fit the data. One peak was centred around an S-phase fraction of 0.9%; the other was centred around 7%. These peaks were separated by a valley at approximately 3%, indicating that the S-phase fraction in NHL can be classified as 'low' (< 3%) or 'high' (> 3%), independent of the median S-phase fraction. The apoptotic fractions were log-normally distributed. The median apoptotic fraction was higher (1.5%) in the 'high' S-phase group than in the 'low' S-phase group . However, there was no significant correlation between the two parameters (P > 0.05).


In vitro, NaPaC inhibited the proliferation of A431 cells (IC50=5 μM). Also, NaPaC decreased the binding of radiolabelled VEGF165 to endothelial cells (IC50=0.2 μM). In vivo, we explored the effects of NaPaC (15 mg kg−1) on A431 xenograft growth starting the drug administration at the time of tumour cell inoculation (early treatment) and 1 week later, when tumours were well established (late treatment). Early treatment was more efficient on tumour inhibition (70% vs control) than late treatment (50% vs control). Early and late NaPaC-treatment increased the aponecrosis in tumour by 70 and 30%, respectively. Whatever treatment, NaPaC inhibited the intratumour endothelial cell density in the same manner. In contrast, vessel area was decreased only when NaPaC was injected early (35%). These results show that NaPaC has a potent inhibitory effect, dependent on treatment outset, on epidermoid carcinoma growth associated with an intratumour microvascular network diminution and an aponecrosis increase. As this drug is nontoxic at efficient dose, it offers interesting perspectives for the therapy of malignant lesions.We investigated the effect of a new dextran derivative, phenylacetate carboxymethyl benzylamide dextran (NaPaC), on epidermoid carcinoma A431 cells secreting a large quantity of angiogenic factor, vascular endothelial growth factor (VEGF).  Angiogenesis, the formation of new blood vessels from established vessels, occurs under a variety of normal and pathological conditions. Also, it is a requisite for tumour growth and metastasis dissemination (in vitro that phenylacetate carboxymethyl benzylamide dextran (NaPaC) inhibited the secretion of growth factors from breast cancer cells and prevented the action of growth factors by interacting with them (165), which is a specific mitogenic factor for endothelial cells. Vascular endothelial growth factor is the best-characterised VEGF-A form the expression of which has been correlated, temporally and/or spatially, with the onset of angiogenesis in a variety of tumours including lung . This calculation leads to MW: 77 607 g mol−1.New dextran derivative, phenylacetate carboxymethyl benzylamide dextran , at 37°C in a 5% CO2-humidified atmosphere.Human epidermoid carcinoma A431 cells and human umbilical vein endothelial cells (HUV-EC) were purchased from American Tissue Culture Collection . They were routinely grown in DMEM , supplemented with 10% FCS, 2 m3) were incubated in 2% FCS–DMEM for 24 h and then treated with NaPaC at different concentrations for 72 h. Then, the cells were washed with phosphate buffer saline (PBS) and incubated with 0.1 ml of MTT (2 mg ml−1) for 4 h.A431 cell growth was assessed using the MTT-microculture tetrazolium assay . BrieflyM125I-VEGF165  in the presence or absence of NaPaC at increasing concentrations (0–24 μM). Incubation was arrested by gently removing the medium and washing the cell monolayer three times with ice-cold binding buffer. The radioactivity bound to cells was measured in gamma counter (LKB 1261 Multigamma) after cell lysis in 0.3 ml of 0.5 N NaOH for 30 min. Nonspecific binding was determined in the presence of an excess (5 nM) of unlabelled VEGF165 . For the Scatchard plot analysis (165 (0–5000 pM) and 7 pM125I-VEGF165 in the presence or absence of NaPaC at IC50. Each curve was analysed according to the Scatchard procedure or by fitting a logistic curve (Graph Pad Software). All analyses were performed twice and carried out in triplicate.HUV-EC and A431 cells were grown until 80% confluence in 24-well tissue culture plates . After an overnight incubation in serum-free medium and two washings with ice-cold binding buffer , cells were incubated at 4°C for 2 h in 0.3 ml of binding buffer containing 7 panalysis , bindingin vivo experiments were carried out with ethical committee approval and met the standards required by the UKCCCR guidelines (5) were inoculated subcutaneously (s.c.) near the right mammary fad pad of 4-week-old athymic nude mice  . Animals were kept in a temperature-controlled room on a 12 : 12 light–dark schedule with food and water ad libitum. Phenylacetate carboxymethyl benzylamide dextran was administrated following two protocols starting at early or late stage of xenograft development. In the first treatment, the administration of NaPaC  begun at the time of A431 cell inoculation: the tumour cells were injected s.c. in 0.1 ml of NaPaC solution. In the control group (n=10), the A431 cells were injected in 0.1 ml of 0.9% NaCl. The drug or 0.9% NaCl solution was administrated twice a week for 5 weeks. In the other protocol (late), the NaPaC treatment started 1 week after cell inoculation when palpable tumours have been developed. It is noteworthy that these tumours were observed in 20 out of 20 animals. Then, mice were arbitrarily placed in control (n=10) and NaPaC-treated group (n=10). Phenylacetate carboxymethyl benzylamide dextran (15 mg kg−1) was injected in 0.1 ml of 0.9% NaCl s.c. near the tumour, twice a week for 5 weeks. Control received 0.1 ml of 0.9% NaCl. Tumour volumes were calculated as previously described , the tumour sections were preincubated with 10% normal goat serum for 1 h at room temperature. Endothelial cells were specifically labelled with GSL-1 isolectin B4 . The GSL-1 lectin binds specifically to galactosyl residues and thus labels the mouse endothelial cells  for 30 min, washed with TBS and incubated with biotinylated rabbit anti-goat immunoglobulins  for 20 min in a moist chamber at room temperature. After three washes with TBS, samples were incubated with streptavidin–biotin peroxidase  for 10 min using 3-amino-9-ethylcarbazole (AEC) chromogen, giving a red staining. Finally, slides were washed in water and counterstained with haematoxylin.Tumour specimens were fixed with a solution of paraformaldehyde (4%) and included into paraffin using standard procedure. Routinely, 5 2 on the picture) (2) was taken into account. The coefficient of variation (SD) was used to assess the variability of counts divided by field number of the same tumour. Mean intratumour endothelial cell numbers per area in the various tumours were compared using Student's t-test. To estimate the area of vessels in tumour section, the lumens bordered with at least one GSL-1-stained endothelial cell were counted using the point-counting grid. The intratumour vessel area was expressed as the ratio of determined counts to total points of grid (96) according to Weibel method  was determined using a point-counting grid over the GSL-1-labelled cells (96 points in the grid corresponding to an area of 1.02 mmpicture) . For eacl method . Thus, vμm) were deparaffinised and rehydrated, then analysed for cell death DNA fragmentation using TumorTACS kit . Intratumour aponecrotic cells were counted using a point-counting grid over the apoptotic cells as described above for endothelial cells. For each tumour section, 10 different fields were selected for analysis.Tumour sections . The NaPaC concentration inducing 50% of maximal inhibition (IC50) was 5 μM.We have recently shown that NaPaC has an antiproliferative effect on various breast cancer cells  and unlabelled VEGF165 at increasing concentrations in the presence (μM NaPaC (IC50). In control conditions (in the absence of NaPaC), two classes of binding sites were observed. The higher affinity class is characterised by a Kd of 100 pM and the lower affinity population by a Kd of 1200 pM. The addition of 0.3 μM (IC50) NaPaC did not significantly affect the affinity of the first class sites, but induced the disappearance of the low-affinity population , NaPaC was able to block VEGF165 binding to high-affinity sites since no specific binding was observed  in a concentration-dependant manner with an IC50 of 0.2 μM , two classes of binding sites as observed by others (Kd of 355 pM and the lower affinity population by a Kd of 1000 pM. The addition of 0.2 μM of NaPaC (IC50) did not significantly affect the affinity of the first class sites, but induced the disappearance of the low-affinity population (data not shown). The disappearance of high-affinity sites was achieved in the presence of drug at a higher concentration (6 μM). Like for A431 cells (above), these experiments clearly showed that NaPaC inhibited the VEGF165 binding to HUV-EC cells probably by forming a complex with the growth factor.Phenylacetate carboxymethyl benzylamide dextran inhibited the binding of VEGFP=0.0067), whereas late administration of the drug inhibited the A431 tumour growth by 50% (P=0.0011). Early administration of NaPaC was not able to affect the A431 tumour uptake. The chronic administration of NaPaC (15 mg kg−1) to A431 xenograft-bearing mice, twice a week for 5 weeks, did not cause signs of toxicity. The body weight of mice was not affected. No diarrhoea, infection, weakness or lethargy was stated. All of the 40 studied mice were alive at the end of treatments.We evaluated the A431 xenograft growth when NaPaC administration begun simultaneously with tumour cell inoculation . In the case of late treatment with NaPaC, the density of aponecrotic cells was increased by 30% compared to control  and the necrotic regions were diminished as compared to early treated tumours   was decreased by 50% (P=0.006) after early NaPaC administration as compared to control (no treated) and 30% (P=0.045) after late treatment as compared to corresponding no treated control  when injected early. Thus, NaPaC, administrated early, is able to affect the endothelial cell number and vessel area whereas NaPaC, injected late, alters only the first parameter.The number of endothelial cells per tumour tissue area  probably because of the high quantity of the secreted endogenous VEGF  treated tumours as well as in control ones. Thus, a first week of A431 xenograft development, in the absence of NaPaC, is sufficient for morphological changes in intratumour vasculature. Interestingly, even 5 weeks NaPaC treatment was not able to affect these changes. The morphological transformations of intratumour vessels were recently described  and since the endothelial cell density was decreased in the same manner in both early and late treated tumours, the most probable is that the difference in tumour growth inhibition was because of changes in intratumour vascular network leading to the increase in tumour cell death observed above. Altogether, our data indicate that A431 xenograft model can be used to study the impact of vascular network in tumour growth and to screen potential antiangiogenic agents.In conclusion, we demonstrated that NaPaC potently inhibits fast-growing epidermoid carcinoma by acting on tumour cells and intratumour endothelial cells whatever the state of xenograft development. Nontoxic at efficient doses, NaPaC provides interesting clues for therapies of solid tumours preventing the vascular network evolution in malignant lesions, thus inhibiting the rapid expansion from small tumours to late-stage tumours. Moreover, its direct inhibitory action on tumour cell proliferation argues for its usefulness in late-stage tumour treatment.


Appreciable insulin occurred in transplants of a hamster and a mouse adrenal tumour. The hormone was measured by immunoassay. In hamster experiments, the weight of tumours grown in animals made mildly diabetic was five times less than those grown in controls. No insulin nor diabetic effect occurred in transplants of a mouse brain or stomach tumour.


The DNA enzyme Dz13, targeted against the oncogene c-Jun, is capable of inhibiting various model tumours in mice albeit in ectopic models of neoplasia. In previous studies using orthotopic models of disease, the inhibitory effects of Dz13 on secondary growth was a direct result of growth inhibition at the primary lesion site. Thus, the direct and genuine effects on metastasis were not gauged. In this study, Dz13 was able to inhibit both locoregional and distal metastasis of tumour cells in mice, in studies where the primary tumours were unaffected due to the late and clinically-mimicking nature of treatment commencement. In addition, the effect of Dz13 against tumours has now been extended to encompass breast and prostate cancer. Dz13 upregulated the matrix metalloproteinase (MMP)-2 and MMP-9, and decreased expression of MT1-MMP (MMP-14) in cultured tumour cells. However, in sections of ectopic tumours treated with Dz13, both MMP-2 and MMP-9 were downregulated. Thus, not only is Dz13 able to inhibit tumour growth at the primary site, but also able to decrease the ability of neoplastic cells to metastasise. These findings further highlight the growing potential of Dz13 as an antineoplastic agent. For ex1311.Dz13 has been shown in ectopic mouse tumour models to reduce the growth of melanoma indirectly via anti-angiogenesis , osteosaThe proteolytic breakdown of proteins of the extracellular matrix (ECM) has long been recognized as a hallmark of invading primary cancer lesions . SeveralOne of the critical proteases involved in cell migration is membrane-type 1 matrix metalloproteinase (MT1-MMP or MMP-14). MT1-MMP degrades extracellular matrix to furnish a path for cells to migrate, sheds cell surface molecules , and activates extracellular signal-regulated protein kinase (ERK), thus enhancing cell migration . ExpressThe activation of the 72 kDa type IV collagenase proMMP-2 (gelatinase A) correlates with increased occurrence of metastases, and leads to a conversion of the 72 kDa pro-MMP species to a 63 kDa zymogen, mediated by MT1-MMP activity at the cell surface . In hepaCleavage of the 92 kDa type IV collagenase proMMP-9 (gelatinase B) results in its activated form, an 82 kDa protein that has been reported to enhance the invasive phenotype of cultured MDA-MB231 cells due to increased capacity of degradation of ECM and transversing basement membrane following activation . In epitThus, this study demonstrates that Dz13 can downregulate MMP-2, MMP-9 and MMP-14 levels in tumour cells, in addition to downregulating its target gene c-Jun, with no effect on the AP-1 transcription factor component (and associate of c-Jun), c-Fos. Moreover, this study seminally demonstrates the direct antimetastatic ability of Dz13 in models of tumour progression.2 incubator, and were ensured to be >95% viable.The human prostate cancer PC3, breast cancer MDA-MB231, osteosarcoma SaOS-2, and osteosarcoma 143B cell lines were from the ATCC , while the osteosarcoma G292 cell line was from D. Thomas . Cells were propagated in α-MEM supplemented with 10% FBS and 1% antibiotic-antimycotic (complete medium). Cell lines were maintained up to 20 passages in a 37°C/5% COThe 34 mer, 10-23 class of deoxyribozymes, Dz13 and Scr (the scrambled sequence control for Dz13) oligonucleotides were synthesised and prepared according to established conditions . DNAzymeTreated cell lysates were immunoblotted as per a published method . BrieflyIHC was performed as before  on parafn = 3, sourced from the Animal Resources Centre, Perth, Australia) were anaesthetised with ketamine (100 mg/kg) and xylazine (10 mg/kg). 2.5 × 105 143B tumor cells in 50% Matrigel  were injected into the inguinal fatpad of 5-week-old female Balb/c nude mice. At the end of the study, the peritoneal cavity was checked for local metastases and the liver and lungs for distal metastases, photographs were taken, and tissues were processed histologically.Prior approval for all animal experimentation was obtained from the St. Vincent's Health Animal Ethics Committee. Female 5-week-old Balb/c nude mice  in saline was administered intraperitoneally 6 weeks later when tumours became palpable. Four weeks later, primary tumours were weighed, and local and lung macrometastases were counted. The peritoneal cavity was checked for local metastases and the lungs for distal metastases, and tumours processed histologically.n = 4 per group) were anaesthetised with ketamine (100 mg/kg) and xylazine (10 mg/kg). 2 × 104 SaOS-2 tumour cells in 50% Matrigel was injected intratibially in a volume of 20 μL in the proximal tibia using a 26G needle and a gentle 'screwing' motion to prevent bone cortex rupture a before [2 [Female 5-week-old Balb/c nude mice (a before . The genefore [2 . A lung efore [2  and sectn = 5 per group) were anaesthetised with ketamine (100 mg/kg) and xylazine (10 mg/kg). Dz13 was mixed with PC3, MDA-MB231 or SJSA-1 tumour cells (1 × 106) at a concentration of 0.4 μM in 50% Matrigel (BD Biosciences) prior to ectopic injection subcutaneously in the midback of 5 week-old Balb/C nu/nu mice. Backflow of the injectate was prevented by retracting the needle post-injection slowly from the injection site. Mice were fed and hydrated ad libitum and monitored twice weekly for tumour development, and then three times weekly when tumours were palpable. Tumour volumes were measured and tumours histologically processed as per published method [Prior approval for use of mice was obtained from the St. Vincent's Health Animal Ethics Committee. Female 5-week-old Balb/c nude mice , prostate (PC3) and breast (MDA-MB231) tumour growths were reduced by Dz13 Fig. . A statiEctopic osteosarcoma G292), prostate (PC3) and breast (MDA-MB231) tumour sections were evaluated by immunohistochemistry for c-Jun and c-Fos (off-target control). c-Jun levels were decreased in all tumours, but c-Fos was not Fig. . Likewis2, prostaIn the future, Dz13 may be a feasible approach to tumour management. Metastasis is the event in tumorigenesis that signals advanced stage disease, and one that is unfortunately frequently not amenable to medical intervention. As such, agents capable of reducing the impact of metastasis are likely to significantly alter the current management strategies for cancers. This is surely the case for OS , where wc-Jun, in the form of the AP-I complex, regulates MMP-9 levels in mammalian cells . This onThus, here we extend Dz13 action on MMPs, specifically showing that MT1-MMP is downregulated when OS cells are treated with Dz13, a novel finding since MT1-MMP was not evaluated in previous Dz13 studies . HoweverThis study is also novel in that it seminally proved the direct effect of Dz13 on tumour metastasis. To test whether Dz13 can directly inhibit tumour metastasis, rather than as a result of primary tumour inhibition as demonstrated before -8, a locIn both the 143B fatpad model and in an orthotopic model for OS, where cells are injected directly into bone , primaryWhile MMPs levels were perturbed in cultured cells, the effects of Dz13 in vivo needed to be tested in a panel of tumour types. The tumours evaluated consisted of prostate, breast and osteosarcoma, though ectopic models were used to examine protein levels via tumour section immunohistochemistry (IHC). When IHC was performed, a consistent decrease in MMP-2 and MMP-9 levels was noted in the Dz13 cohort of animals compared to both the saline-treated and Scr-treated tumours. Thus, while in vitro results suggest that MMP levels may fluctuate and even increase via activation, in vivo results showed a consistent decrease in MMPs by Dz13. The discrepancy could be due to the fact that in culture, cells were exposed to a very potent Dz13 onslaught in a 2-D configuration, while in vivo, cells were protected by the Matrigel and the 3-D nature of the injected 'colony' of cells, and treatment was protracted over 5 weeks. However, what was consistent was the finding that both in vitro and in vivo, levels of MT1-MMP were reduced in tumour cells treated with Dz13 but not the scrambled control.In addition, in vivo, significant decrease in c-Jun levels were noted for all ectopic tumours treated with Dz13 but not when cells were treated with the Scr control oligonucleotide. In vitro, cells have to be serum-starved prior to growth induction (and c-Jun elevation) before Dz13 shows it effects , but in For Dz13, the present set of results further highlight the inherent potential of this molecule. Not only is it able to control tumour at the primary site, but also at the secondary site as well. As for other tumours, and particularly in OS, while the primary tumour may be removed in a good number of cases, it is the metastases which become life-threatening . FurtherThe authors declare that they have no competing interests.CRD conceived the project and designed the experiments. CRD and MLT performed the studies. PFMC provided clinical foresight to the studies. CRD prepared, and MLT and PFMC edited the manuscript. CRD gave approval for the final version to be submitted.


The response of 6 human colorectal tumour xenografts to 7 cytotoxic agents have been established. Tumour responses have been quantified by growth inhibition, and the time taken for 3H-thymidine fractional incorporation (TFI) to recover to the control value after treatment. The chemosensitivity of each tumour line to a spectrum of agents was individual, and no pattern of response which would allow prediction of individual agent efficacy was apparent. Cyclophosphamide, methyl-CCNU and 5-fluorouracil produced marked growth inhibition in individual tumour lines, whereas actinomycin-D, cis-dichlorodiammine platinum, doxorubicin and pentamethylmelamine showed little activity. Data presented agree with clinical evaluation for single-agent therapy. The uptake and incorporation of radiolabelled 5-fluorouracil into 4 tumour lines is reported. No marked differences between 3 FU-insensitive lines and 1 sensitive line have been observed.


An anti-tumour agent 5,6-dimethylxanthenone-4-acetic acid  induced nitric oxide synthase (NOS) in the tumour, spleen, thymus and small intestine, but not in the lung, liver, kidney, heart or skeletal muscle in B6D2F1 mice bearing subcutaneous colon 38 tumours. This pattern of induction is distinct from that caused by agents such as endotoxin, muramyl dipeptide or Corynebacterium parvum. The induction of NOS (iNOS) in the tumour was more persistent  than in other tissues . Immunohistochemical staining suggested that iNOS was located in macrophages and endothelial cells within and around the tumour. Treatment with 5,6-MeXAA also caused substantial increases in plasma nitrite and nitrate (NOx) concentrations that peaked at 8-12 h after 5,6-MeXAA. The increase in plasma NOx was prevented by a NOS inhibitor N-iminoethyl-L-ornithine (L-NIO), indicating that it was due to enhanced production of NO. Tumour-bearing mice were more responsive than controls to 5,6-MeXAA both in their plasma NOx increase and in their lower maximally tolerated dose. L-NIO was unable to prevent the complete tumour necrosis and regression caused by 5,6-MeXAA at a dose that substantially inhibited the increase of plasma NOx. In conclusion, the experimental anti-tumour agent 5,6-MeXAA induced NO synthesis in tumour-associated macrophages and in immunologically active tissues in parallel with its effects on tumour growth. The experiments with a non-selective NOS inhibitor L-NIO, however, suggest that NO is not a significant component in the mechanism of the anti-tumour action of 5,6-MeXAA in this particular model.


Recent studies have shown that elevated levels of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1) in breast cancer correlate with an increased risk of a reduced relapse-free survival time and shortened overall survival times. Urokinase PA and PAI-1 are independent prognostic indicators for breast cancer. The fact that plasminogen activators are indispensable for tube formation of microvascular cells and that they may induce angiogenesis in vitro strongly suggests a role for uPA and PAI-1 in tumour neovascularisation. Because macrophages and tumour cells produce uPA, we postulate a close collaboration between tumour cells and tumour-associated macrophages in angiogenesis. To investigate how uPA levels and macrophage counts in tumour tissue correlate with angiogenesis, we counted microvessels and determined uPA levels and macrophage content in 42 primary invasive breast carcinomas. Using light microscopy, we highlighted the vessels by staining their endothelium cells immunocytochemically for CD31 and factor VIII and the macrophages for CD68. After obtaining tumour tissue extracts, we determined the uPA and PAI-1 levels by ELISA. A positive correlation between microvessel density, vascular invasion, uPA level, macrophage content and proliferation rate was found.


Growth of transplanted rat tumours was retarded and in some cases completely suppressed when cells were injected subcutaneously in admixture with double stranded RNA (ds-RNA). This response required intimate contact between ds-RNA and tumour cells and systemic treatment with the agent failed to prevent progressive growth of a range of rat tumours. Direct cytotoxic effects of ds-RNA may contribute to tumour suppression since the compound was cytotoxic in vitro for cultured tumour cells. The involvement of host factors is suggested, however, by the in vivo tests showing variations in susceptibility to ds-RNA mediated tumour suppression similar to that previously observed with bacterial adjuvants.


We found that overexpression of ephrin-B2 markedly decreased tumour growth in a mouse xenograft model. Immunohistochemical staining showed that ephrin-B2 transfectants produced higher tumour microvessel density and lower tumour cell proliferation than did parental or vector-transfected control cells. Using 51Cr-labelled red blood cells (RBCs) to determine the functional blood volume in tumours, we demonstrated that tumours from ephrin-B2-transfected cells had significantly decreased blood volume compared with tumours from parental or vector-transfected control cells. Evaluation of in vitro parameters of cell cycle mediators demonstrated no alteration in the cell cycle. Although ephrin-B2 transfection increased tumour vessel density, the decrease in blood perfusion suggests that these vessels may be ‘dysfunctional’. We conclude that overexpression of ephrin-B2 suppresses tumour cell growth and vascular function in this in vivo colon cancer model.Eph receptor tyrosine kinases (RTKs) and their membrane-bound ligands, the ephrins, are essential for embryonic vascular development. Recently, it has been demonstrated that overexpression of specific Ephs and ephrins is associated with a poor prognosis in human tumours. Our group has shown that EphB and the ephrin-B subfamilies are coexpressed in human colorectal cancer, and ephrin-B2 is expressed at higher levels in human colorectal cancer than in adjacent normal mucosa. As the Eph/ephrin system is involved in embryologic vasculogenesis and ephrin-B2 is expressed ubiquitously in all colon cancers studied in our laboratory, we hypothesised that overexpression of ephrin-B2 in colon cancer cells may induce tumour angiogenesis and increase tumour growth. To investigate this hypothesis, we stably transfected KM12L4 human colon cancer cells with ephrin-B2 to study its effect on tumour growth  The largest subfamily of receptor tyrosine kinases (RTKs), the Eph family, and their ligands (ephrins), play important roles in physiologic and pathologic vascular processes. Both the Ephs (receptors) and ephrins (ligands) are divided into two groups, A and B subfamilies . The binEph RTKs and their ephrin ligands play important roles in embryonic vascular development. Disruption of specific Eph receptors and ligands (including ephrin-B2) leads to defective vessel remodelling, organisation, and sprouting resulting in embryonic death .Our group has shown that EphB and the ephrin-B subfamilies are co-expressed in human colorectal cancer, and ephrin-B2 is expressed at higher levels in human colorectal cancer than in adjacent normal mucosa . Murine 10T1/2 cells were grown in basal Eagle's medium supplemented with 10% FBS, and 2 U ml−1 penicillin–streptomycin. HUVECs were cultured on 0.5% gelatin  in modified MEM supplemented with 15% FBS, 10 ng ml−1 recombinant human basic fibroblast growth factor, penicillin–streptomycin, vitamins, sodium pyruvate, L-glutamine, and nonessential amino acids. All cells were grown at 37°C in 5% CO2 and 95% air.The KM12L4, KM20, KM23, KM12C, and KM12SM human colon cancer cell lines were provided by Dr IJ Fidler  , rat anti-mouse CD31/PECAM-1 antibody and peroxidase-conjugated rat anti-mouse immunoglobulin G1 (IgG1) from Pharmingen ; mouse antiproliferating cell nuclear antigen (PCNA) clone PC10 DAKO A/S from DAKO Corp. ; and peroxidase-conjugated goat anti-rat IgG (H+L) and peroxidase-conjugated rat anti-mouse IgG2a from Serotec Harlan Bioproducts for Science, Inc. .7–108 cells from colon cancer cell lines or nonmalignant cell lines growing in culture by using a FastTrack mRNA isolation kit . Northern blot analysis was performed as previously described , and 0.1% sodium dodecyl sulphate. Autoradiography was then performed.Polyadenylated mRNA was extracted from 10Ephrin-B2 was subcloned into the Kpn I/Not I sites of pcDNA3 (Invitrogen), a eukaryotic expression vector driven by the human cytomegalovirus promoter and containing a neomycin-resistance gene. Completeness and orientation of the insert were determined by means of restriction enzyme analyses and DNA sequencing .μg ml−1) was used to select for clones. Individual colonies were expanded and examined for ephrin-B2 expression by Northern blot analysis. Clones with a high expression of exogenous ephrin-B2 were pooled to establish an additional experimental cell line, the ephrin-B2 S pool.The ephrin-B2 sense plasmid as well as vector plasmid alone (pcDNA3) were transfected into KM12L4 cells utilising the FuGENE 6 transfection system, according to the manufacturer's protocol . At 48 h after transfection, medium containing G418  of KM12L4, pcDNA3-transfected KM12L4, or ephrin-B2-sense transfected KM12L4 (S2 or S pool) cells were injected subcutaneously in the right upper flank of 10 mice each. Prior to injection, a trypan blue exclusion test was carried out to assure that cell viability was greater than 80%. Tumour growth was measured every other day. Tumour volume was calculated using the formula: volume=(diameter2 × length)/2. All animal studies were conducted according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) of M.D. Anderson Cancer Center and were performed in accordance with the UKCCCR guidelines for the welfare of animals in experimental neoplasia  were obtained from the National Cancer Institute's Animal Production Area , acclimated for 1 week, caged in groups of five, and fed a diet of animal chow and water μm thick), mounted on positively charged Superfrost slides , and allowed to dry overnight at room temperature. Sections were deparaffinised in xylene followed by 100, 95, and 80% ethanol and rehydrated in phosphate-buffered saline . These sections were used for haematoxylin and eosin (H&E) staining and detection of PCNA. Sections analysed for PCNA were microwaved for 5 min to increase antigen retrieval before staining, and stained as described below.Paraffin-embedded tumours were sectioned (4–6 μm thick), mounted on positively charged Superfrost slides, and air-dried for 30 min. Tissues were fixed in cold acetone (5 min), 1 : 1 acetone/chloroform (5 min), and acetone (5 min) and then washed with PBS three times for 3 min each. These sections were used for CD31 staining. Specimens were then incubated with 3% hydrogen peroxide in PBS (v v−1) for 12 min at room temperature to block endogenous peroxidase. Sections were washed three times for 3 min each with PBS (pH 7.5) and incubated for 20 min at room temperature in a protein-blocking solution consisting of PBS supplemented with 1% normal goat serum and 5% normal horse serum.Tumours frozen in OCT solution were sectioned ] and PCNA (peroxidase-conjugated rat anti-mouse IgG2a) were diluted 1 : 200 and 1 : 100, respectively, in protein-blocking solution. After incubation with the secondary antibody for 1 h at room temperature, the samples were washed and incubated with stable diaminobenzidine  substrate. Staining was monitored under a bright field microscope, and the reaction was stopped by washing with distilled water. For the CD31 staining, sections were counterstained with Gill's No. 3 haematoxylin  for 15 s and mounted with Universal Mount (Research Genetics). Control specimens were treated with a similar procedure except that the primary antibody was omitted.Ephrin-B2-sense-transfected cells (S2) and vector-transfected cells (KM12L4/pcDNA3) were seeded in the wells of four-chamber slides (Fisher Scientific Co.) and cells were grown until 80% confluent. The cells were fixed with cold acetone  for 10 min and washed with PBS twice for 5 min each. Immunofluorescent staining was performed as previously described  mounted on a Zeiss universal microscope  and Optimas Image Analysis software  installed on a Compaq computer with Pentium chip, a frame grabber, an optical disk storage system, and a Sony Mavigraph UP-D7000 digital color printer . Microvessels were quantified according to the method described by To quantify tumour microvessel density and tumour cell proliferation, tumour microvessels (stained for CD31) and PCNA-positive cells were evaluated by light microscopy and counted in three random 0.159 mm51Cr) (Amersham) was added to label the RBCs. The labelling reaction was carried out in a 37°C water bath with shaking for 2 h. The 51Cr-labelled RBCs were then washed twice with PBS for 15 min each time and resuspended in HBSS. A 0.2-ml suspension of 51Cr-labelled RBCs was injected intravenously into the tail vein of tumour-bearing mice ((n=10/group), ∼0.05 mCi per mouse). At 10 min after injection of 51Cr-labelled RBCs, 10 μl of blood was collected from the tail vein of each mouse for radioactive count reading. The mice were then killed, the tumours were removed, and radioactive counts of the tumours were recorded with the Packard Auto-Gamma counter . The blood volume in the tumours was calculated using the formula: blood volume (μl g−1)=tumour radioactive count (cpm g−1)/blood radioactive count (cpm μl−1).Five nude mice were killed and their blood was harvested in heparinised syringes (approximately 1 ml per mouse) for radiolabelling. Red blood cells (RBCs) were centrifuged at 1000 rpm for 5 min and resuspended in 1 ml of PBS containing Hank's balanced salt solution (HBSS) (Life Technologies). In all, 1 mCi of radiochromium (51Cr labelling) was compared using the Mann–Whitney U test for nonparametric data. All analyses were carried out using InStat Statistical Software  with P⩽0.05 considered to be significant.Tumour weights, tumour volumes, tumour microvessel densities, and numbers of PCNA-positive cells were compared between groups by using one-way analysis of variance (ANOVA). Functional blood volume in tumours , as expected (data not shown). In addition, to confirm that the transfected ephrin-B2 protein product localised to the cell surface, we performed immunofluorescent staining on cells ol cells . As expe weights  than didP<0.0001, Figure 3AP<0.0001)  had significantly higher tumour vessel counts compared to controls . Tumours from ephrin-B2-sense-transfected cells had a significantly lower blood volume per gram of tissue than did tumours from pcDNA3-transfected cells (P=0.0288) (Overexpression of ephrin-B2 led to an increase in tumour angiogenesis (vessel count by CD31 staining) but a decrease in tumour size and tumour cell proliferation (PCNA staining). This apparent paradox could be explained by the development of an inefficient blood supply in the ephrin-B2-overexpressing tumours. Therefore, we hypothesised that despite more vessels being present in the ephrin-B2-overexpressing tumours, these tumours have a decreased functional vascular volume/flow due to inefficient blood supply. To investigate this hypothesis, we used =0.0288) .Ephrins consist of two classes: ephrin-As, which bind to the membrane via a glycosylphosphatidylinositol anchor, and ephrin-Bs, which interact with their receptors through the transmembrane cytoplasmic domain. Ephrins/Ephs are essential mediators in the development of the nervous  and cardAmong the ephrin family members, ephrin-B2 is critically important for embryologic vascular development. Ephrin-B2 knockout mice resulted in severe vascular developmental defects during embryogenesis  increased tumour cell death, (2) decreased tumour cell proliferation, and (3) alterations in angiogenesis that can affect both processes. Using Western blot analysis, we determined the protein expression profiles for regulators of the cell cycle  and of apoptosis  in ephrin-B2-transfected and control cells. These studies were carried out to determine whether ephrin-B2 transfection altered any of these parameters that could directly affect tumour growth in vivo. None of these proteins was altered by ephrin-B2 transfection (data not shown). Furthermore, cell cycle analysis by fluorescence-activated cell sorting showed that the cell cycle distribution of ephrin-B2 transfectants was the same as that of control cells (data not shown). In addition, an in vitro analysis of cell proliferation/number (MTT assay) likewise did not show any differences among groups (data not shown). Therefore, based on these analyses, we concluded that the decrease in tumour growth observed in the current study was not due to changes in the cell cycle or tumour cell apoptosis. We therefore hypothesised that despite the increase in microvessel density, these vessels were dysfunctional. To evaluate the ‘functional’ tumour vascular volume, we injected 51Cr-labelled RBCs and harvested blood and tumours to obtain the relative radioactivity. Tumours from ephrin-B2-transfected cells had a significantly smaller tumour blood volume despite having an increase in microvessel density. These data demonstrated that overexpression of ephrin-B2 can increase tumour microvessel density but that these vessels may be ‘dysfunctional’. The fact that these tumour vessels were dysfunctional is similar to the findings of Jain and co-workers (We transfected the full-length ephrin-B2 cDNA into the human colon cancer cell line KM12L4 and found that the -workers . Our obs51Cr-labelled RBCs suggested that tumour vessel count does not always correlate with the ‘functional status’ of the vasculature. This further questions the value of vessel counts as a measure of angiogenic or antiantiangiogenic activity (In summary, our study demonstrated two novel findings with potentially important implications. The first was that overexpression of ephrin-B2 on tumour cells can markedly decrease the growth of SQ colon cancer xenografts. The second interesting finding was that this decrease in tumour growth occurred despite an increase in tumour vessel counts. Our experiment with


We have developed a new technique to measure in vivo tumour tissue fluid transport parameters (hydraulic conductivity and compliance) that influence the systemic and intratumoral delivery of therapeutic agents. An infusion needle approximating a point source was constructed to produce a radially symmetrical fluid source in the centre of human tumours in immunodeficient mice. At constant flow, the pressure gradient generated in the tumour by the infusion of fluid  was measured as a function of the radial position with micropipettes connected to a servo-null system. To evaluate whether the fluid infused was reabsorbed by blood vessels, infusions were also performed after circulatory arrest. In the colon adenocarcinoma LS174T with a spherically symmetrical distribution of Evans blue-albumin, the median hydraulic conductivity in vivo and after circulatory arrest at a flow rate of 0.1 microl min(-1) was, respectively, 1.7x10(-7) and 2.3x10(-7) cm2 mmHg(-1) s. Compliance estimates were 35 microl mmHg(-1) in vivo, and 100 microl mmHg(-1) after circulatory arrest. In the sarcoma HSTS 26T, hydraulic conductivity and compliance were not calculated because of the asymmetric distribution of the fluid infused. The technique will be helpful in identifying strategies to improve the intratumoral and systemic delivery of gene targeting vectors and other therapeutic agents.


Non-small-cell lung cancer (NSCLC) is an aggressive disease in which vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) are implicated in tumour growth, tumour resistance to radiation and chemotherapy, and disease relapse. We have investigated the anti-tumoural effects of BMS-690514, an inhibitor of both vascular endothelial growth factor receptor (VEGFR) and epidermal growth factor receptor (EGFR) signalling pathways, as a single agent and in combination with ionising radiation (IR) on several NSCLC cell lines.in vitro using clonogenic assay and in vivo using nude mice.Radiosensitisation of several NSCLC cell lines by BMS-690514 was assessed In vitro studies showed that BMS-690514 alone decreases clonogenic survival of NSCLC cells lines but no potential enhancement of IR response was observed in the combination. In tumour-bearing mice, BMS-690514 alone inhibits the growth of NSCLC xenografts, including the T790M mutation-harbouring H1975 tumour. The concomitant combination of BMS-690514 and radiation did not increase mice survival in comparison with treatment with IR alone. In contrast, BMS-690514 markedly enhances the anti-tumour effect of radiation in a sequential manner on H1299 and H1975 xenografts. Immunohistochemistry revealed a qualitative reduction in vessel area after administrations of BMS-690514, compared with vehicle-treated controls, suggesting that revascularisation may explain the schedule dependency of the tumour-growth delay observed.The results of association with radiation show that BMS-690514 may be a successful adjuvant to clinical radiotherapy. These findings are of translational importance because the clinical benefits of anti-EGFR and anti-VEGFR therapy might be schedule dependent. Lung cancer is a major health problem worldwide and is the leading cause of cancer-related death for men and women .Inhibiting the epidermal growth factor (EGF) pathway is another promising approach for improving the anti-tumoural activity of radiation. A pivotal study reported a benefit in overall survival and locoregional control in patients with localised head and neck cancer who were treated with both radiation and cetuximab, a monoclonal antibody against epidermal growth factor receptor (EGFR), over patients treated with radiation alone  expression .Preclinical studies have demonstrated enhanced radiation-induced cell kill when anti-angiogenic therapy is combined with radiotherapy , H1650 cells , and H1975 cells (EGFR L858R/T790M and WT p53) were maintained in RPMI 1640 with GlutaMAX supplemented with 10% foetal calf serum and antibiotics (as above). Cultures were maintained in a humidified incubator at 37°C and 5% CO2. For in vitro experiments, BMS-690514 was dissolved in dimethyl sulphoxide at 50 μmol l−1 as a stock solution and stored at −20°C. For in vivo studies, BMS-690514 was prepared fresh before each administration. It was dissolved in 1,2-propanediol and Tween 80 to a final 1,2-propanediol concentration of 40% and Tween 80 concentration of 10%.A549 cells (wild-type (WT) EGFR and p53) were grown in F12-K medium containing L-glutamine, supplemented with 10% foetal calf serum, 100 U ml−1, cells were washed and exposed to radiation at a dose ranging from 2 to 6 Gy using 200-kV X-rays, and then cells were trypsinised, counted, and seeded for colony formation in 35-mm dishes at 50–1000 cells per dish. After incubation intervals of 14 days, colonies were stained with crystal violet and manually counted. All colonies of 50 or more cells were then counted. The survival fraction (SF) was estimated according to the following formula: SF=number of colonies formed/number of cells seeded × plating efficiency of the control group. All experiments were performed in triplicate.First, a clonogenic assay was performed without irradiation and with various doses of BMS-690514 to demonstrate dose response and calculate the concentration of BMS-690514 to inhibit 50% of cells (IC50). To investigate the effect of BMS-690514 on the response of cell lines to radiation, a standard clonogenic assay was performed. Survival after radiation exposure was defined as the ability of cells to maintain clonogenic capacity and form colonies. Briefly, after incubation intervals of 12 h with BMS-690514 at a dose ranging from 20 to 500 nmol lIn vivo experiments were carried out at the Institut Gustave Roussy under Animal Care license no 94-076-11 (Ministère de l’Agriculture). Female athymic nude mice (6–8 weeks of age) purchased from Janvier CERT  were used. A549, H1975, and H1299 cells were collected in exponential growth phase and ∼3 × 106 cells were injected s.c. into the flank area of 6- to 8-week-old female athymic nude mice on day 0. When tumours reached the appropriate size, mice were randomised into groups of six mice each and treated.Three separate animal experiments were performed:3 were randomly assigned to receive BMS-690514 (30 mg kg−1 day−1) or vehicle . Treatment was administered once daily by oral gavage for 5 days.(a) BMS-690514 alone: Mice bearing tumours with a volume of 75–150 mm3 were randomised into groups of six to orally receive either vehicle or BMS-690514 (30 mg kg−1 per os quaque die (every day) × 5, × 1 week) once daily for the duration of the experiment. BMS-690514 or vehicle was administered with or without irradiation (6 Gy in a single administration on day 3). Localised irradiation was administered at a dose rate of 0.85 Gy min−1 using mouse jigs.(b) BMS-690514+IR: For BMS-690514 and radiation studies, mice bearing established tumours of a volume of 75–150 mm(c) IR+adjuvant BMS-690514: For H1299 and H1975 xenografts, BMS-690514 was given the first day after IR at a dose of 6 Gy, and from day 2 to day 12 according to a sequential schedule.Mice were weighed, and tumour size was measured twice a week with an electronic caliper. Individual mice were followed up over 30 days after the beginning of the treatment. Tumour volume was estimated from two-dimensional tumour measurements using the following formula: Vt/Vo ratio (Vt as the mean tumour volume on a given day during the treatment and Vo as the mean tumour volume at the beginning of the treatment).In each group (six mice per group), the relative tumour volume was expressed as the The experimental end point was taken as the time for the tumour to reach a relative tumour volume of five times that at the initiation of therapy (RTV5). When tumours reached RTV5, mice were killed. Overall survival was calculated using the Kaplan–Meier method. Growth delay was calculated by subtracting the average time for control tumours to grow five-fold in volume from the time required for treated tumours to increase in volume by the same amount from the first day of treatment.μm thick) were prepared. After xylene treatment and rehydration, immunohistochemistry of anti-CD34-positive murine endothelial cells  was performed. Heat-induced epitope retrieval was achieved with Tris–EDTA (pH 8) at 98°C for 40 min. Endogenous peroxidase activity was quenched by treatment with 3% H2O2 for 10 min. The sections were placed in coverplates  and incubated with blocking serum Biogenex wash buffer  for 10 min. This step was followed by incubation with anti-mouse CD34 (1:20 dilution) diluted in blocking serum (1:10 dilution) for 1 h. Slides were then incubated with a rabbit anti-rat (1:400)  diluted in blocking serum (1:10 dilution). The following step was performed using a Rabbit PowerVision kit  for 20 min and diaminobenzidine for 10 min. Slides were counterstained with Mayer's hematoxylin and mounted with Pertex. Tumour necrosis was assessed by light microscopy. The number of microvessels per field was scored by averaging five field counts of three individual tumours for each group.Histological assessment was carried out to evaluate the effects of BMS-690514 on tumour vasculature. Three animals were killed after the 3-day treatment with BMS-690514. Tumours were excised and, after fixing them in Finefix , paraffin sections -harbouring cell line (H1975), a mutation that confers resistance against erlotinib . Clonogenic survival was not different in the four NSCLC cell lines treated with BMS-690514, radiation, or both.No potential se of 20  or 500 nP=0.02), H1299 tumour-bearing mice (P=0.02), and H1975 tumour-bearing mice (P=0.1), compared with mice treated with vehicle. The time taken to reaching a relative A549 tumour volume equivalent to five times that at the start of the treatment (RTV5) was 40±12 days with BMS-690514 compared with 22±11 days with vehicle (P=0.02). Similar data were observed for H1299 tumour-bearing mice: RTV5 was 17±3.5 days with BMS-690514 compared with 9±1.2 days with vehicle (P=0.02). RTV5 was 23±5 days in H1975 tumour-bearing mice administered BMS-690514 compared with 14±4 days for mice treated with vehicle (P=0.15).Continuous oral dosing of A549, H1299, and H1975 tumour-bearing mice with BMS-690514 for 5 days resulted in tumour growth inhibition. To evaluate the potent interaction between BMS-690514 and radiation, tumour-bearing mice were treated with either BMS-690514 or radiation, or both, in concomitant schedule.−1 BMS-690514 was given orally once a day from day 1 to day 5 and 6-Gy radiation was administered at day 3. No increase in survival was observed in mice treated with both treatments , exhibited a significant increase in survival , compared with mice treated with radiation alone  or BMS-690514 alone . Furthermore, the association leads to an increase in time to reach RTV5 as compared with mice treated with BM-690514 alone or radiation alone  exhibited a significant increase in survival  compared with mice treated with radiation alone  or BMS-690514 alone .While on a concurrent schedule, no enhancement of radiation was observed; BMS-690514 markedly enhances the anti-tumour effect of radiation in a sequential manner . H1299 xon alone . The asson alone . Sequent−1 day−1), the proportion of tumour vessels seemed to be lesser than that in vehicle-treated controls and a qualitative reduction in CD34-positive vessel area was observed.As shown in −1 day−1) is well tolerated in athymic mice , the proportion of abnormal tumour vessels seemed to be lesser than that in vehicle-treated controls and a qualitative reduction in CD34-positive vessel area was observed (The radiopotentiating effect of BMS-690514 was reduced when BMS-690514 was administered concomitantly with radiation as compared with a sequential schedule. After 3 days of BMS-690514 administration (30 mg kgobserved . Hence, The type of cell lines tested may also influence the sequence dependency of the findings with concurrent irradiation, as comparisons between models strongly and weakly expressing EGFR show that EGFR ‘strong’ cell lines exhibit both increased tumour growth delay after exposure to IR and a dual EGFR–VEGFR2 inhibition with paradoxical superiority of concomitant versus adjuvant sequencing in EGFR ‘weak’ cell lines (in vivo tumour growth delay in NSCLC models (A preclinical evaluation of the combination of ionising radiation and 4 h after the last radiation dose showed that a vascular disrupting agent, ZD6126, combined with the anti-EGFR Gefitinib did enhance Altogether, the sequence dependency effects of the combination of anti-vascular agents and anti-EGFR inhibitors on ionising radiation may differ depending on models and drugs. However, there is consistency regarding the beneficial effect of adjuvant treatment. Our data are in line with these findings. Given the fact that this type of scheduling offers minimal toxicity hazard compared with concurrent treatment, adjuvant treatment should be the optimal treatment in a clinical setting.In conclusion, these data show that inhibition of both the VEGF and EGFR pathways demonstrates efficacy in NSCLC xenografts and that combining such agents with radiation could improve the anti-tumour activity of radiation alone. However, scheduling seems to be an important factor to be taken into account to establish optimal effect. This study supports the development of BMS-690514 in NSCLC as an adjuvant to radiotherapy in a clinical.