We assessed the efficacy of DLI and DC in generating potent graft vs neuroblastoma tumour (GVT) reactions following allogeneic BMT.Mounting evidence points to the efficacy of donor lymphocyte infusion (DLI) and immunisation with tumour-pulsed dendritic cells (DC) in generating graft a) Neuro-2a tumours were grafted with allogeneic (H2Kb) T-cell-depleted bone marrow cells. Tumour-pulsed donor DC (DCNeuro2a) were inoculated (on day +7) in conjunction with donor (H2Kb) and haploidentical (H2Ka/b) lymphocytes.Mice bearing congenic (H2KNeuro2a and lymphocytes devoid of graft vs host (GVH) activity (H2Ka/b). In variance, the efficacy of DC-mediated immunomodulation was diminished by severe graft vs host disease (GVHD), showing mechanistic dissociation and antagonising potential to GVT.Murine Neuro-2a cells elicit immune reactions as efficient as B lymphoma in major histocompatibility complex antigen-disparate mice. Lymphopenia induced by conditioning facilitates GVT, and transition to adaptive immunity is enhanced by simultaneous infusion of and DCThe GVHD is not a prerequisite to induce GVT reactivity after allogeneic BMT, but is rather detrimental to induction of anti-tumour immunity by DC-mediated immunomodulation. Simultaneous inoculation of tumour-pulsed donor DC and DLI synergise in stimulation of potent GVT reactions to the extent of eradication of established NB tumours. In variance from previous studies aiming to decipher the mechanism of interaction between antigen-presenting cells and lymphocytes , C57BL/6 , B6.SJL-Ptprca Pepcb/BoyJ , inbred A:C57BL/6 F1 chimeras  and non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice purchased from Jackson Laboratories . Mice were housed in a barrier facility in accordance with the guidelines of the Institutional Animal Care and Use Committee.Mice used in this study were A/J (H2Ka) and A20 B-cell lymphoma (H2Kd) were obtained from the American Type Culture Collection . Cells were cultured to maximum 12 passages as previously described . Tumour growth was measured with a caliper and the volume (mm3) was calculated according to: (width2 × length × 0.4).Neuro-2a cells murine NB (H2KWhole bone marrow cells (wBMCs) were harvested from the femurs and tibia of donors, and low-density cells were collected as previously described . Immunomμm mesh and dispersed into single-cell suspensions in PBS  in low-LPS RPMI 1640 (<10 pg ml–1) supplemented with 10% FBS, 1% L-glutamine, 1% sodium pyruvate, 1% α-MEM non-essential amino acids, 0.1% Hepes buffer and 1% Pen/Strep . The medium was supplemented with 50 ng ml–1 mouse recombinant (mr) granulocyte macrophage colony-stimulating factor (mrGM-CSF) and 10 ng ml–1 interleukin-4 (mrIL-4) on intermittent days . Naïve DCs were incubated for additional 24 h in DC medium without growth factors. Pulsing with tumour antigens was performed by co-incubation of DC with tumour lysate at an approximate DC/tumour cell ratio of 3 : 1  and cultured  at a rate of 106 rad minμl M199, centrifuged over 1.5 ml lymphocyte separation media (Cedarlane), and red blood cells were lysed. Nucleated cells were incubated for 45 min at 4°C with phycoerythrin (PE)-anti-H2Kb  and fluorescein isothiocyanate (FITC)-anti-H2Kk mAb cross-reactive with H2Ka . Minor antigen disparity was assed using CD45.1-PE and CD45.2-FITC antibodies (eBioscience). T cells were quantified using CD4-allophycocyanin and CD8-FITC antibodies . Measurements were performed with a Vantage SE flow cytometer . Positive staining was determined on a log scale, normalised with control cells stained with isotype control mAb.Blood was collected in heparinised serum vials in 200 2 asphyxiation, and were fixed in ice-cold PBS containing 1.5% fresh paraformaldehyde for 2 h at 0–4°C. Tissues were embedded in OCT , frozen in isopentane suspended in liquid nitrogen, sectioned (3–6 μm) with a Cryotome  and stained with haematoxylin and eosin.Skin and liver were collected from mice killed by COvs host disease was assessed using a semiquantitative clinical scale including weight loss, posture (hyperkeratosis of the foot pads impairs movement), activity, diffuse erythema (particularly of the ear) or dermatitis, and diarrhoea. GVHD was validated by histology in haematoxylin and eosin sections of the ear and liver according to: 0 – no infiltration, 1 – scarce infiltrates, 2 – patchy infiltration and 3 – diffuse infiltration with deterioration of tissue structure. The modified score for the liver (Graft μM 5-(and-6-)-carboxyfluorescein diacetate succinimidyl ester . Triplicate cultures were harvested after 3–5 days, evaluated for CFSE dilution by flow cytometry and data were quantified using the ModFit software . In some cases the cells were exposed to mitogenic stimulation with 5 μM concanavalin A .Splenocytes were layered in plastic dishes for 45 min and non-adherent cells were labelled with 2.5 5 Neuro-2a target cells for 7 h at 37°C in 150 μl at 1 : 10–1 : 100 target/effector ratios. Cytolysis was quantified by lactate dehydrogenase (eBioscience) release and normalised for background values.Effector splenocytes harvested from naïve mice and chimeras were lysed and passed through wool mesh to enrich for T lymphocytes (∼70%). These cells were incubated with 5 × 10post hoc Scheffe t-test and significance was considered at P<0.05.Data are presented as means±s.d. for each experimental protocol. Results in each experimental group were evaluated for reproducibility by linear regression of duplicate measurements. Differences between the experimental protocols were estimated with a b) were immunised twice at 3-day interval, and lymphocytes were co-incubated with target cells (a) were gradually killed at decreasing target/effector ratios and were comparable to lysis of A20 . Therefore, the Neuro-2a cell line elicits effective immune responses comparable to an immunogenic cell line  T-cell-depleted BMCs and on day +7 were inoculated with 106 donor DCs (subcutaneous) and 2 × 107 donor (H2Kb) and F1 (H2Ka/b) splenocytes , resulting in remarkable superior efficacy of F1-DLI . Furthermore, early allogeneic DLI induced high-grade GVHD , whereas infusion of F1 splenocytes alone neither affect Neuro-2a growth P<0.001,  accompanb) and haploidentical splenocytes (H2Ka/b). The stimulatory effect of DC was evident from marked proliferative responses of lymphocytes to mitogenic stimulation at the experimental end point (4 weeks after BMT). Inoculation of tumour-pulsed DC alone enhanced the cycling rates of donor lymphocytes derived from the bone marrow of the chimeras, with a less accentuated effect after simultaneous administration of DC and F1 lymphocytes , emphasising effective DC-mediated immune activation in vivo as a basis for tumour growth suppression.Donor DC present antigens in the context of compatible MHC to both allogeneic . Thus, DC administration has a stimulatory effect over lymphoid reconstitution; however, adoptive transfer of F1 lymphocytes in conjunction with BMCs does not suppress tumour growth.Given that the combination of F1 lymphocytes and DC suppressed tumour growth, we reasoned that fostering immune reconstitution by adoptive transfer of lymphocytes at the time of transplantation might provide additional benefit . SequentNeuro2a. As F1 lymphocytes are not detected in the lymphoid organs of recipients at 4 weeks after transplantation  and was apparently unaffected by F1 lymphocyte infusion on day +7 . Qualitaantation . These d3). Although tumour-pulsed DC suppressed the growth of Neuro-2a cells under all conditions . Consistently, the profile of complete regression of tumours exceeding 60 mm3 mirrored the variations in tumour size (The relative efficacy of the various protocols was determined by tumour size at the experimental end point and eradication of established tumours (>60 mm P<0.01, , adoptivour size , reinforNeuroblastoma is a potential target of GVT reactivity attained by allogeneic BMT and subsequent immunomodulation. Murine NB expresses MHC class II antigens (Transplant procedures associated with lymphopenia are generally associated with increased susceptibility to development of GVT reactions , and thein vitro. These data corroborate previous reports of potent vaccination following syngeneic BMT (Professional processing and presentation of tumour antigens by DC elicits anti-tumour reactivity and causes complete regression of established NB tumours. Antigen-specific responsiveness is demonstrated by vigorous proliferative responses of lymphocytes upon rechallenge with tumour-pulsed DC neic BMT .ex vivo expanded donor CD4+ T cells mediated reactivity against disseminated recurrent NB (In contrast to our initial assumption that early reinforcement of lymphoid reconstitution at the time of transplantation will favour GVT reactivity, simultaneous administration of F1 lymphocytes and donor DC converged to suppress tumour growth. This differential behaviour reflects distinct mechanisms of action, given that F1 lymphocytes engraft transiently and are undetected at 4 weeks after transplantation . In variEffective GVT reactivity attained by transplantation of allogeneic T-cell-depleted BMC is dissociated from the detrimental GVH disease . Both GVIn summary, NB is a solid tumour effectively attacked by immune reactions, which can be reinforced by post-transplant immunomodulation. As BMT becomes a standard procedure in high-risk patients, allogeneic transplants have the significant advantage of generating potent GVT reactions. Anti-tumour reactions are promptly initiated by DC at relatively early stages after transplantation under conditions of partial reconstitution of the T-cell compartment without causing GVHD, and can be augmented by DLI. These synergistic measures are significant in the competition against an aggressive and fast growing tumour such as NB, underlining the importance of optimisation of the details of the transplant procedure.


Murine monoclonal antibodies directed against tumour associated antigens are potentially useful in tumour diagnosis and therapy. However, all the antigens they recognise may be heterogeneously expressed on tumours and this may allow escape of cells from therapy if a single monoclonal antibody is used. One approach is to use combinations of monoclonal antibodies recognising complementary cell surface antigens. A flow cytometric method which allows accurate quantitation of the intensity of staining and the percentage of fresh primary tumour cells binding a series of monoclonal antibodies has therefore been developed. This allows calculations as the number of drug molecules which could be potentially delivered by each monoclonal antibody and the optimal combination of antibodies which should be used. Monoclonal antibodies recognising Y hapten (C14), CEA  and 791T-p72 antigen (791T/36) have been screened as a possible combination for colorectal cancer. There was inter-tumour variation in the binding of all the monoclonal antibodies although combinations could reduce or abrogate this problem. A combination of the monoclonal antibodies C14, 228, 791T/36 and 161 would recognise 100% of tumours. Sixty per cent of tumours bound all four antibodies, 78% any three, 90% any two and 100% any one antibody. There was also intra-tumour variation in the number of tumour cells per lesion that were recognised, the best monoclonal antibody, 161, stained a mean of 59% of cells per tumour whereas the anti-cytokeratin monoclonal antibody stained a mean of 74% of cells per tumour. An increased intensity of staining of tumour membranes was observed when a combination of C14 and 228 was used compared to binding of individual antibodies. Furthermore there was still no significant binding to normal colon membranes. Combinations of monoclonal antibodies which recognise a high percentage of tumours are likely to be necessary for monoclonal antibody drug targeting to prevent tumour recurrence and/or metastases.


A fluorescent probe has been employed to study the status of a tumour associated protease, guanidinobenzoatase, in frozen sections of human tumours obtained from the head and neck regions. The results indicate that in vivo a naturally occurring inhibitor of guanidinobenzoatase effectively controls the activity of this enzyme on the majority of cells in a tumour mass. This inhibitor can be artificially displaced by formaldehyde treatment of the frozen sections and this treatment reveals the extent of latent enzyme in the section. In the frozen sections it was noticed that at the advancing edges of the tumour mass, the tumour cells possessed uninhibited guanidinobenzoatase, an enzyme known to degrade the link peptide between cells and fibronectin. It was shown that a synthetic inhibitor of guanidinobenzoatase selectively inhibited the guanidinobenzoatase of the tumour cells at the advancing edge of the tumour mass. It is suggested that the guanidinobenzoatase on cells at the leading edge of the tumour mass plays an important role in the invasion of adjacent host tissue. This synthetic inhibitor of guanidinobenzoatase has no inhibitory action on other trypsin-like enzymes and might therefore be of value in limiting the growth of the tumour mass in vivo.


Rabbits were injected with VX2 cancer cells into the left thigh or tibia, and given indomethacin 1-16 mg/kg daily starting on the day before tumour implantation or 7, 14 or 21 days after implantation. Indomethacin at 2 mg/kg and above from before tumour implantation reduced osteoclast proliferation and the amount of prostaglandin-like material extracted from homogenates of excised tumours, but the inhibition of bone destruction in vivo was significant only with indomethacin at 4 mg/kg and above. Indomethacin at 8 mg/kg reduced osteoclast proliferation and bone destruction, but the effect was statistically significant only when given within 7 days of inoculation with the tumour. The place of indomethacin and other inhibitors of prostaglandin synthesis has not yet been established in the management of patients with skeletal metastases. Drug administration might need to be started at the time of diagnosis and removal of the primary tumour, rather than when skeletal metastases are evident.


Experiments were performed in amelanotic melanoma A-MEL 3 implanted in a transparent dorsal skinfold chamber of the hamster. Starting at day 0 after tumour cell implantation, animals were treated daily with the anti-angiogenic compound SU5416 (25 mg kg bw−1) or vehicle (control) only. Functional vessel density, diameter of microvessels and red blood cell velocity were visualised by both orthogonal polarisation spectral™ imaging and fluorescence microscopy and analysed using a digital image system. The morphological and functional properties of the tumour microvasculature could be clearly identified by orthogonal polarisation spectral™ imaging. Data for functional vessel density correlated excellently with data obtained by fluroescence microscopy . Correlation parameters for diameter of microvessels and red blood cell velocity were similar . Treatment with SU5416 reduced tumour angiogenesis. At day 3 and 6 after tumour cell implantation, respectively, functional vessel density was 4.8±2.1 and 87.2±10.2 cm−1 compared to values of control animals of 66.6±10.1 and 147.4±13.2 cm−1, respectively. In addition to the inhibition of tumour angiogenesis, tumour growth and the development of metastasis was strongly reduced in SU5416 treated animals. This new approach enables non-invasive, repeated and quantitative assessment of tumour vascular network and the effects of antiangiogenic treatment on tumour vasculature in vivo. Thus, quantification of tumour angiogenesis can be used to more accurately classify and monitor tumour biologic characteristics, and to explore aggressiveness of tumours.Tumour angiogenesis plays a key role in tumour growth, formation of metastasis, detection and treatment of malignant tumours. Recent investigations provided increasing evidence that quantitative analysis of tumour angiogenesis is an indispensable prerequisite for developing novel treatment strategies such as anti-angiogenic and antivascular treatment options. Therefore, it was our aim to establish and validate a new and versatile imaging technique, that is orthogonal polarisation spectral™ imaging, allowing for non-invasive quantitative imaging of tumour angiogenesis British Journal of Cancer (2002) 86, 1622–1627. DOI: 10.1038/sj/bjc/6600318www.bjcancer.comCancer Research UK© 2002  Animals bearing solid tumours were treated with anti-angiogenic therapy using the synthetic FLK-1 kinase inhibitor SU5416  to block the signal transduction pathway through the VEGF receptor  and xylazine . Dorsal skin was shaven and chemically depilated . The dorsal skinfold chamber . The ethical guidelines that were followed meet the standards required by the the UKCCCR ‘Guidelines for the welfare of animals in experimental neoplasia’ (n=8) were treated daily by intraperitoneal injection of SU5416 twice  dissolved in DMSO at a final concentration of 25 mg kg−1 body weight. This dosage of SU5416 has been proven to be effective previously in several different animal species (n=8) received the DMSO only.After tumour cell implantation, animals were assigned randomly to two groups. Starting on the day of tumour implantation, animals  for visualisation of microvessels. For fluorescence microscopy a 20-fold water immersion objective  with a total magnification of 533× was used. A 150 W halogen lamp  in combination with a fluorescence filter block  was used to visualise FITC-Dextran. Fluorescence images were captured using a charge-coupled device video camera . The images from both devices (Cytoscan A/R and fluorescence microscopy) were recorded on S-VHS video tape  for later off-line analysis.Tumour angiogenesis was quantified at days 3, 6, 9 after tumour implantation. For the experiments, the awake animals were immobilised in a plexiglass tube and the chamber preparation was attached to a microscope stage, which was computer controlled to allow for repeated scanning of identical segments of microvessels. To take advantage of the computer controlled microscope functions the OPS imaging probe was attached to the shaft of the microscope. The plexiglas stage was modified so that there were two different pairs of holes, with which the stage could be attached to the motorised plate. One pair of holes aligned the chamber under the fluorescence microscope and the other set aligned the chamber under the OPS imaging probe. Moving the stage from one pair of holes to the other, the identical region of interest could be observed with the two systems. OPS imaging was using the new Cytoscan E-II instrument   were measured.Six regions of interest (ROI), three from the centre of the tumour and three from the periphery of the tumour, were randomly selected for the assessment of tumour angiogenesis. Analysis of microcirculatory parameters using OPS imaging as well as fluorescence microscopy were performed off-line from the videotapes by digital image analysis system . Functional vessel density (fvd) as parameter of tumour angiogenesis (one per cm), defined as the total length of perfused microvessels per unit area of observation, was quantified  were injected subcutaneously in the dorsal skin of hamsters .In addition to morphological features, OPS imaging offers the potential to monitor noninvasively and continuously the microhaemodynamics of tumours. Therefore, we compared diameter and red blood velocity of tumour microvessels with data obtained by intravital fluorescence microscopy. The correlations were excellent for measurements of red blood cell velocity , 147.4±13.2 and 87.2±10.2 cm−1 at day 6 (P<0.05), and 119.7±7.2 and 106.1±14.7 cm−1 at day 9 (−1 at day 3, 0.08±0.02 and 0.11±0.04 mm s−1 at day 6, and 0.09±0.02 and 0.07±0.02 mm s−1 at day 9 after tumour cell implantation, for control and SU5416 treated animals, respectively. Similarly, no effect of SU5416 treatment on microvascular diameter could be noted. Microvessel diameter was 17.6±2.1 and 12.6±3.8 μm at day 3, 14.5±1.4 and 15.2±1.5 μm at day 6, and 16.6±0.4 and 15.3±2.5 μm at day 9 after tumour cell implantation, for control and SU5416 treated animals, respectively.Treatment of tumours with SU5416 reduced the formation of new vessels. The functional vessel density of tumours monitored by OPS for control and SU5416 treated animals, respectively, was 66.6±10.1 and 4.8±2.1 cmat day 9 . With re3 for control vs 1.2±0.2 cm3 for anti-angiogenic treated animals. There was no difference in animal body weight over the whole period of observation. Axillary metastases became palpable in all control animals between day 9 and day 11 after tumour cell implantation. In the SU5416 group, only in one animal axillary metastasis formation was palpable at day 19 after tumour cell implantation. Total volume of axillary metastasis at the end of the investigation was 1.09±0.31 vs 0.05±0.05 cm3 for control and SU5416 treated animals, respectively.Exponential tumour growth was observed in all tumours of control animals. In addition to the inhibition of new vessel formation, daily treatment of animals with SU5416 resulted in a significant delay of tumour growth . At the in vivo. We have established and validated this new technique for the tumour microvasculature to allow for characterisation of angiogenesis and the effects of anti-angiogenic treatment of tumours. Simultaneous measurements by OPS imaging were compared with intravital fluorescence microscopy investigations of tumour angiogenesis, red blood velocity and vessel diameter. For microvessel density, a well established parameter for tumour angiogenesis (OPS imaging is a new technique to visualise microvessels ogenesis . The addogenesis . FurtherThe present study supports SU5416 as a potent synthetic inhibitor of angiogenesis by blocking the VEGF/Flk-1 signal transduction pathway. This compound, which is currently under investigation in Phase III clinical studies for the treatment of human tumours, has been shown to have anti-angiogenic properties and broad antitumour effects leading to a reduction of tumour growth (in vivo. The small size of the probe facilitates its use as a non-invasive diagnostic tool in both experimental and clinical settings to evaluate and monitor angiogenesis/angiogenic activity of tumours known to impact the prognosis of the disease. The ability to obtain high contrast images of the vasculature using reflected light will allow for a non-invasive and repetetive quantitative measurement of the distinct morpholgical specifities of the new vasculature such as vascular volume, surface and density as well as quantitative information about the functional characteristics of vasculature with parameters like microvessel density, and vascular dynamics which are both important requirements for angiogenesis assays (OPS imaging technology provides a new method for the assessment of tumour microvessels


Salmonella serovar typhimurium recombinant for cytosine deaminase (TAPET-CD) has been documented in experimental settings. The present data demonstrate that in vivo19F-magnetic resonance spectroscopy measurements allow the outcome prediction of this prokaryotic-based therapy, demonstrating the necessity of non-invasive real-time imaging techniques for treatment monitoring.The potential utility of tumour-selective 5-fluorouracil treatment using attenuated  Preclinical animal investigations and patient studies suggest that trapping of 5-FU in the tumour correlates with response and may provide prognostic information on treatment outcome (Positron emission tomography and (nuclear) magnetic resonance imaging (MRI) have been introduced to evaluate the biological characteristics of tumours and to monitor the efficacy of treatment on an individual basis. Fluor-containing chemotherapeutics, such as 5-fluorouracil (5-FU) and its metabolites, can be detected non-invasively in tumour and liver tissues using Clostridium and attenuated Salmonella, can be used to transfer CD selectively to the rodent tumour microenvironment, resulting in antitumour effects  enzyme, which converts the nontoxic 5-fluorocytosine (5-FC) into the cytostatic 5-FU, in a number of experimental settings. Among these, we and others have demonstrated that recombinant bacteria such as in vivo19F-MRS to predict the success of the TAPET-CD/5-FC cancer therapy system and to evaluate it as a tool to allow individualised treatment.The aim of the present study was to investigate the value of 6 cells per 100 μl) subcutaneously at the lower flank of 8- to 10-week-old female NMRI nu/nu mice. Treatment and 19F-MRS measurements started when tumour volumes reached >300 mm3. Tumours were measured in three orthogonal dimensions with a calliper and volumes were calculated according to the formula ABC × π/6.HCT116 human colorectal carcinoma cells were xenografted (1.5 × 106 bacteria were injected intravenously (tail vein). Six days after TAPET-CD administration, 5-FC was daily administered intraperitoneally in one group of animals (n=12), whereas a control group (n=4) received saline according to the same schedule.TAPET-CD , in detail described by in vivo19F-MRS experiments were performed in a 4.7 T BIOSPEC horizontal magnet (Bruker), equipped with a double-tuned (19F-1H) surface coil of 1 cm diameter, as described previously  in a 1 : 5000 dilution was used as primary antibody. After overnight incubation of the sonicated lysates with 5-FC (10 mg ml−1), the conversion into 5-FU was analysed by TLC on silica gel plates eluted with butanol. 5-Fluorocytosine and 5-FU were loaded as reference. Histological evaluation of hypoxia and necrosis was performed on paraffin-embedded tumour pieces after administration of the exogenous hypoxia marker pimonidazole. Staining and evaluation of the sections were performed as described previously  and activity (thin-layer chromatography (TLC)) of CD, according to In vitro19F-MRS analysis was performed on perchloric acid extracts using a AMX360 (8.4 T) spectrometer , the others as ‘non-responders’ (3 out of 12). Linear regression analysis showed a significant tumour growth delay for the ‘responders’ compared to the ‘non-responders’  and control saline-treated group , as witnessed from the interaction between time and group  (9 cfu g−1 tumour tissue). In addition, Western blotting on tumour extracts did not reveal differences in CD protein expression (in vitro19F-MRS (nd group . RepreseP=0.044) . This wapression . To exclpression  left. Th19F-MRS . Interes19F-MRS  right.in vivo19F-MRS. More importantly, we demonstrate the need for sequential non-invasive 19F-MRS imaging as a tool for TAPET-CD/5-FC therapy prediction. Given the fact that a pilot clinical trial with TAPET-CD, in which two out of three refractory cancer patients maintained stable disease when functional CD was produced within the tumour . These results showed that prodrug diffusion to the bacterial colonisation sites in both groups is not impaired. As no differences were seen in bacterial colonisation and CD protein expression, but significance was obtained in growth delay between ‘responders’ and ‘non-responders’, we reasoned that the observed difference was probably owing to either the presence of an inhibitory factor in the tumours of the ‘non-responders’, or to the lack of factors needed for CD enzyme activity. The catalytic activity of CD is shown to be dependent on Fe2+ or other divalent metal ions that function as cofactor. Although it might be possible that these cofactors are present in varying amounts among tumours, this appeared not to be limiting in our study (data not shown). Alternatively, low intratumoural pH or high endogenous cytosine concentrations that compete with 5-FC for the active site might reduce CD activity. However, as TLC experiments revealed that adding fresh growth medium to the tumour extracts of non-responders resulted in 5-FC/5-FU conversion, our data point towards the absence of yet other necessary factors. From our results, it is apparent that in a subgroup of tumours, the absence of these necessary nutrients for efficient CD enzymatic activity can result in a lack of antitumour efficacy. Therefore, our results stress the importance of non-invasive real-time imaging tools as 19F-MRS for individualised treatment as it allows the prediction of antitumour efficacy when using TAPET-CD/5-FC therapy or related treatments.Tumours in which a 5-FU signal could be detected using


Meta-tetrahydroxyphenylchlorin (m-THPC) and 5-aminolaevulinic acid (5-ALA) are two second-generation photosensitizers which are currently under investigation for photodynamic therapy (PDT) and photodynamic diagnosis (PDD). So far, the experience with these photosensitizers for use within brain tumours is limited. We examined the distribution and retention of 14C-labelled m-THPC and [14C]5-ALA in the rat C6 glioma brain tumour model. After intraperitoneal injection of m-THPC ; 0.16 mg ml(-1) m-THPC; 0.3 mg kg(-1)), the following activities were found after 36 h: brain tumour 223,664 d.p.m. g(-1), brain contralateral to the tumour side 2567 d.p.m. g(-1), liver 369,959 d.p.m. g(-1) and skin 55,197 d.p.m. g(-1); 100,000 d.p.m. corresponding to 0.22 microg of m-THPC. After 7 days, the concentration of m-THPC decreased to 76,277 d.p.m. g(-1) in tumour and 635 d.p.m. g(-1) in brain. The radioactivity after intravenous administration of [14C]5-ALA ; 40 mg ml(-1); 120 mg kg(-1)) increased within 15 min  in tumour, 17,427 d.p.m. g(-1) in brain); after 8 h only a small amount (3653 d.p.m. g(-1) in tumour) remained. Brain adjacent to the tumour was also found to have a higher uptake of 5-ALA. This study provides basic information for the use of m-THPC and 5-ALA in brain tumours. Because of the different pharmacokinetic and toxicological profile, we recommend m-THPC for PDT and 5-ALA for PDD. Clinical trials now have to prove the superior phototoxic properties of these second-generation photosensitizers.


With progressive growth of syngeneic sarcomata in rats there was a rise in serum levels of lysozyme which correlated with their immunogenicity and their macrophage content. By an examination of lymph/blood differences in normal and in tumour bearing rats and of the production of lysozyme by cells obtained from the tumours and maintained in vitro, it is apparent that the macrophages resident in a tumour mass make a massive contribution to the elevation in serum lysozyme concentrations. Tumour cells did not release detectable lysozyme activity. Tumour amputation led to a rapid fall in lysozyme levels. Irradiation of the host rats abolished the lysozyme response and the subsequent development of metastases in these rats was associated with a rise in serum lysozyme. The serum concentration of this enzyme reflects the macrophage content of a tumour mass and the draining lymph nodes. We conclude that under well defined conditions serum lysozyme activity may be a useful marker of macrophage mediated host responses to a tumour.


The ratio of circulating MMP-2/TIMP-1 was also significantly higher in episodes of tumour control vs tumour progression and prior to treatment (P⩽0.0001). We conclude that serum MMP-2 appears to reflect tumour resorption, while serum TIMP-1 may mirror tumour expansion.Connective tissue turnover plays a prominent role in tumour growth and metastasis. We followed serum levels of seven connective tissue parameters in 37 patients with colorectal cancer metastatic to the liver prior to and during chemotherapy. Serum samples with episodes of tumour control ( Matrix metalloproteinases (MMPs) are proteolytic enzymes that play a central role in benign and malignant matrix remodelling . MMP-2 aCEA and CA 19-9 have been previously shown to predict tumour growth in patients with liver metastasis treated by chemotherapy (−2) followed by a 24-h infusion of 5-fluorouracil (2600 mg m−2). One cycle comprised six weekly infusions followed by 2 weeks of rest. A total of 16 patients received additional biweekly oxaliplatin (85 mg m−2) and three patients additional weekly irinotecan (80 mg m−2)  with colorectal carcinoma metastatic to the liver. Primary carcinoma was confirmed histologically. Histological confirmation was also obtained for synchronous liver metastasis. In case of metachronous liver metastasis, histological confirmation was only pursued when imaging techniques  of the abdomen or MRT of the liver) did not show clear results. Patients received first-line chemotherapy, consisting of a weekly 1–2 h infusion of folinic acid (500 mg m mg m−2) . Treatme mg m−2) .−1 (s.d. 5.5%) for MMP-2 and 620 ng ml−1 (s.d. 4.9%) for TIMP-1.Sera were obtained at 8 weekly intervals, along with CT, stored frozen at −80°C and analysed for six circulating connective tissue markers in a single run. Serum collagen IV and VI, tenascin-C, MMP-2, the MMP-9/TIMP-1 complex and free TIMP-1 were measured on the Bayer Immuno 1™ Analyzer using fluorescein- and alkaline-phosphatase-labelled monoclonal antibodies to the target antigen . All analyses were performed using SPSSWIN 9.0.For descriptive analysis all sera referring to ‘prior to treatment’, ‘tumour control’ and ‘tumour progression’ were grouped together. For confirmatory analysis, values were averaged for each patient to yield only one value per category. With only four of the patients who contributed values for initial tumour status showing progression, confirmatory analysis was restricted to the comparison of ‘prior to treatment P<0.01 and P<0.05, resp.). Serum MMP-2 was elevated in 9% (two out of 23) prior to treatment, increasing to 35% (40 out of 112) in tumour control and decreasing to 12% (two out of 16) with progression   in 57% (13 out of 23), decreasing to 36% (40 out of 112) in episodes with tumour control and increasing to 81% (13 out of 16) in episodes with progression (This is the first report of the course of serum connective tissue markers in patients undergoing chemotherapy for colorectal carcinoma metastatic to the liver. Among the six tested markers, only MMP-2 and TIMP-1 correlated with the response to chemotherapy. Changes in elimination do not explain the differences in MMP and TIMP-1 serum levels, since during chemotherapy both serum creatinine and parameters of cholestasis remained unaltered.The increase of MMP-2 and the decrease of TIMP-1 under tumour control is unexpected, since a common assumption is that enhanced activity of MMP-2, the classical basement membrane collagenase, leads to tumour growth and metastasis, while TIMP-1 as a crucial inhibitor of many MMPs should rather curtail tumour expansion . HoweverTherefore, therapies of advanced tumours that are based on unselective MMP inhibitors as a supplement to chemotherapy may be of little use, if not counterproductive. This assumption is supported by the disappointing outcome of several recent clinical studies using such agents (


Comparative biodistribution studies of similar sized DFM and F(ab́)2 and TFM and IgG, radiolabelled with both 131I and 90Y have been described previously using the human colorectal tumour LS174T nude mouse xenograft model  Br J Cancer74: 1397–1405). In this study quantitative estimates of radiation distribution and RIT in the xenograft model provided more insight into selecting the most suitable combination for future RIT. Radiation doses were significantly higher in all tissues when antibodies were labelled with 90Y. Major contributing organs were the kidneys, liver and spleen. The extremely high absorbed dose to the kidneys on injection of 90Y-labelled DFM and F(ab́)2 as a result of accumulation of the radiometal would result in extremely high toxicity. These combinations are clearly unsuitable for RIT. Cumulative dose of 90Y-TFM to the kidney was 3 times lower than the divalent forms but still twice as high as for 90Y-IgG. TFM clears faster from the blood than IgG, producing higher tumour to blood ratios. Therefore when considering only the tumour to blood ratios of the total absorbed dose, the data suggests that TFM would be the most suitable candidate. However, when corrected for equitoxic blood levels, doses to normal tissues for TFM were approximately twice the level of IgG, producing a two-fold increase in the overall tumour to normal tissue ratio. In addition RIT revealed that for a similar level of toxicity and half the administered activity, 90Y-IgG produced a greater therapeutic response. This suggests that the most promising A5B7 antibody form with the radionuclide 90Y may be IgG. Dosimetry analysis revealed that the tumour to normal tissue ratios were greater for all 131I-labelled antibodies. This suggests that 131I may be a more suitable radionuclide for RIT, in terms of lower toxicity to normal tissues. The highest tumour to blood dose and tumour to normal tissue ratio at equitoxic blood levels was 131I-labelled DFM, suggesting that 131I-DFM may be best combination of antibody and radionuclide for A5B7. The dosimetry estimates were in agreement with RIT results in that twice the activity of 131I-DFM must be administered to produce a similar therapeutic effect as 131I-TFM. The toxicity in this therapy experiment was minimal and further experiments at higher doses are required to observe if there would be any advantage of a higher initial dose rate for 131I-DFM. © 1999 Cancer Research CampaignWe have been investigating the use of cross-linked divalent (DFM) and trivalent (TFM) versions of the anti-carcinoembryonic antigen (CEA) monoclonal antibody A5B7 as possible alternatives to the parent forms (IgG and F(ab́)


Information on primary tumour size, status of the pelvic lymph nodes, histological type and macroscopic tumour appearance, as well as age and sex, was available at presentation for 394 patients in the Co-operative Urological Cancer Group's prospective randomised trial for T3 cancer of the urinary bladder. An apparently significant prognostic effect of age and sex was shown to be entirely consistent with the effect of natural mortality. Primary tumour size was found to be the single most powerful prognostic factor (P = 0.002), followed by nodal status (P = 0.02). These factors do not act independently. Multivariate analysis showed that 75% of the effect of all the six variables and their first order interactions could be explained by a single prognostic grouping based on tumour size and nodal status only. Three levels for this grouping are proposed: node-negative small tumour, node-negative moderate tumour and either node-positive or large tumour. The 3-year survival probabilities for the three prognostic groups were 85.7% (95% CI 57.2 and 96.4%), 60.3% (48.0 and 71.5%) and 33.3% (23.5 and 44.8%) respectively.


We have examined the biological properties of CEA6, a human carcinoembryonic antigen (CEA)-specific single-chain Fv (scFv) isolated by phage display, and five related clones derived by affinity maturation and selected for improved off-rate (Koff). All clones bind strongly and specifically to CEA-positive human tumours by immunocytochemistry and show negligible cross-reactivity with normal colon. Flow cytometry of scFv on human liver cells indicates a shift in fine epitope specificity resulting from mutagenesis. All monomeric scFv have been radioiodinated, retaining effectively full binding activity. A single intravenous injection into nude mice bearing human colon tumour xenografts confirms tumour targeting in all cases. As reported in other studies, the kidney is the main route of elimination of scFv at early time points. Tumour binding of the parental antibody CEA6 consistently gives the highest tumour-blood ratios at 24 h (mean 16:1). Clone TO6D11, which has a sevenfold reduced Koff relative to CEA6, showed no difference in tumour uptake at 24 h but persisted at the tumour site for longer than CEA6. This study demonstrates a possible correlation between binding affinity and tumour residence time when examined in this model.


To determine the mechanisms associated with loss of androgen dependency and disease progression in prostate cancer (PCa), we investigated the relationship between the androgen receptor (AR) and mTOR pathways and the impact of inhibiting both pathways in androgen-dependent and castration-resistant PCa models.3, animals were grouped and injected subcutaneous with vehicle, our novel anti-androgen/androgen synthesis inhibitor, VN/124-1, bicalutamide, and everolimus. Tumour volumes were measured biweekly. The PSA and protein analyses were performed after completion of the treatment.Androgen-dependent (LNCaP) and castration-resistant PCa (HP-LNCaP) cells were grown as tumours in SCID mice. Once tumours reached 500 mmThe addition of everolimus to bicalutamide treatment of resistant tumours significantly reduced tumour growth rates and tumour volumes. Anti-androgen treatment also increased protein expression of multiple signal transduction pathways earlier than vehicle-treated control xenografts. VN/124-1 plus everolimus acted in concert to reduce tumour growth rates in our castration-resistant xenograft model.This study suggests that dual inhibition of AR and mTOR in castration-resistant xenograft models can restore sensitivity of tumours to anti-androgen therapy. Furthermore, after bicalutamide failure, dual inhibition with VN/124-1 and everolimus was the most effective treatment. The progression to castration-resistant prostrate cancer (CRPC) requires the ability to overcome two major roadblocks, the initial survival of prostate cancer (PCa) cells during the acute onset of androgen deprivation and the ability to proliferate in a castrate-independent manner. During castrate-resistant progression, PCa cells adapt to alternative cellular signalling to survive in an androgen-depleted environment.The lack of successful treatments for CRPC is, in part, a reflection of the poor understanding of the mechanisms and pathways that lead to increased cell survival after androgen ablation. Several mechanisms and pathways have been proposed to explain castrate resistance. The two main mechanisms of progression are those that involve the androgen receptor (AR) and those that bypass the AR. However, these pathways are not mutually exclusive, and cross-talk between the AR and signal transduction pathways (STPs) in CRPC has been demonstrated , 2005b.We and others have shown that PCa cells can become hypersensitive to androgens as a means of circumventing androgen blockade . Hypersevice versa  solution, and Dulbecco's phosphate-buffered saline were obtained from Invitrogen . Regular serum and charcoal-stripped fetal bovine serum (FBS) were obtained from Hyclone . The C4-2B cells were kindly supplied by Dr Yun Qiu . Dimethyl sulfoxide (DMSO), 3--2,5-diphenyltetrazolium bromide (MTT), and Tween 20 were obtained from Sigma Chemicals Company . The ECL chemiluminescence kit and Hybond-ECL nitrocellulose membranes were purchased from Amersham Biosciences . Antibodies against p-Akt (Ser-473 and Thr-308), p-mTOR (Ser2448 and Ser2481), p-MAPK, IGFR1, p-P70S6K, p-PS6, immobilised Akt, and β-actin were purchased from Cell Signaling Technology, Beverly, MA, USA. The p-HER2 was purchased from Upstate  and AR (SC-441) was obtained from Santa Cruz Biotechnology . The total PSA ELISA kit was purchased from Diagnostic Systems Laboratories in Webster . Bicalutamide (Casodex) was kindly provided by Dr Elizabeth Anderson . Everolimus (42-O-(2-hydroxy) ethyl-rapamycin) (RAD-001) was kindly provided by Dr D Evans , and dissolved in 100% DMSO for cell culture application. Novel compound VN/124-1 (3β-hydroxy-17-(1H-benzimidazol-1-yl)androsta-5,16-diene) was designed and synthesised by one of us, Dr Vincent CO Njar , 0.25% trypsin-EDTA (1 mmol lin vivo-derived cell line from LNCaP cells (C4-2B) were maintained in T-Medium with 10% FBS and 1% penicillin/streptomycin (Androgen-dependent PCa cells derived from metastatic lymph node of a PCa patient (LNCaP) were routinely maintained in RPMI 1640 medium with 10% FBS and 1% penicillin/streptomycin solution  of protein from each sample were separated on a denaturing polyacrylamide gel, transferred to nitrocellulose membrane, and western blotting was carried as previously described  were determined using log–log curve fit to the standard curve.Tissue lysates and serum were loaded in the total PSA ELISA Kit  in triplicates to determine PSA concentrations. The assay standards, controls, and samples were incubated in 96-well plates following the manufacturer's instructions. Each original experimental sample was assayed in triplicate. The PSA values  at 2.0 × 107 cells per ml. Each mouse received a subcutaneous (s.c.) inoculation at one site per flank with 100 μl of cell suspension. Tumour formation varied between 4 and 8 weeks by cell line, although on tumours reaching 500 mm3, mice were grouped by 5 or 10 with equal tumour volumes. Tumour size was measured twice weekly with calipers, and tumour volume was calculated by the formula 4/3πr12 × r2, where r1 is the smaller radius. Treatments varied between 3 and 7 weeks by experiment. All mice were killed by decapitation under anaesthesia, and the trunk blood was collected for PSA analysis. Tumours were also excised, cleaned, weighed, and stored in −80°C for additional analysis.All animal studies were performed according to the guidelines and approval of the Animal Care Committee of the University of Maryland School of Medicine. Male SCID mice 4–6 weeks of age were obtained from the National Cancer Institute-Frederick Cancer Research Center . The mice were housed in a pathogen-free environment under controlled conditions of light and humidity and received food and water P-values were two sided. Mixed-effects models were used to compare average tumour volume on pre-specified time points. The Tukey–Kramer method was used to adjust for multiple comparisons.Data on tumour volume and weight were analysed separately. Growth curve models were used to summarise the pattern of response over treatment duration. The tumour growth rate (the slope of regression line) was compared between pre-specified treatment groups. The mean tumour volume was modelled as a combination of fixed effects shared by all subjects and random effects unique to a particular animal. We used random effects for both intercept and slope at the animal level. The proportions of tumours that shrunk below a threshold during treatment were compared using Fisher's exact test. The treatment groups were compared with one another at 0.05 level of statistical significance. All reported −10 M in LP-LNCaP cells and inhibited by 50% at 10−9 M in HP-LNCaP cells. The HP-LNCaP cell proliferation was stimulated in a biphasic manner, although the dose response curves were shifted 3-log to the left  and treated with bicalutamide (100 mg kg−1 q.d. s.c.) or everolimus (2.5 mg kg−1 every 2 weeks p.o.). When tumours had doubled in volume (tumour progression) on day 7, animals in the bicalutamide group were subgrouped (n=10) by equal tumour volume and continued on anti-androgen only or administered everolimus (2.5 mg kg−1 every 2 weeks p.o.) in addition to bicalutamide (100 mg kg−1 q.d. s.c.).The effect of treatment with AR and mTOR inhibitors was compared with the addition of an mTOR inhibitor to bicalutamide therapy, as tumours progressed on the anti-androgens. A total of 40 male SCID mice were inoculated with LNCaP cells and monitored weekly for tumour formation. When tumours reached 500 mmAs seen in P=0.66. The average tumour volume on day 7 was not different between mice that stayed on bicalutamide and the group administered everolimus in addition to bicalutamide; P=0.59. By day 26, the growth rates between bicalutamide plus everolimus and control (P<0.0001) or bicalutamide (P<0.0001) were significantly reduced. Animals treated with everolimus alone, compared with everolimus added to bicalutamide, also had statistically different growth rates (P=0.04).Animals in the control and bicalutamide group were monitored for over 26 days, the everolimus group for over 33 days, and mice treated with bicalutamide plus everolimus were studied for 65 days. Linear mixed-effects models were used to analyse the data. Random effects for intercept and slope at the tumour level nested within a mouse were used to fit the model. Data on tumour volume were log transformed to satisfy the assumptions of the linear mixed-effects models. There were no differences in average tumour volume on day 0 across treatment groups; The results suggest that in the LNCaP xenograft model, the addition of everolimus to bicalutamide treatment of resistant tumours was more effective than either drug alone, and restored the sensitivity of tumours to anti-androgen therapy.n=15) and were treated with vehicle (control) or bicalutamide (100 mg kg−1 q.d. s.c.), and tumours were measured weekly (n=5) with equal tumour volumes were isolated at three different time points  and killed. Tumours were homogenised and prepared for protein analysis as described in Materials and Methods.We sought to determine the molecular impact of the anti-androgen treatment over time in a CRPC model. For this investigation, we analysed tumours collected at the same time points. A total of 30 male SCID mice were inoculated with HP-LNCaP cells. Measurable tumours formed in ∼4 weeks after inoculation. Mice were then grouped  and treatment was initiated. The six groups included control, VN/124-1, bicalutamide, everolimus, bicalutamide plus everolimus, and VN/124-1 plus everolimus.A total of 30 male SCID mice were inoculated with HP-LNCaP cells. Approximately 4 weeks later, on tumour formation, mice were divided into six groups (−1 b.i.d. s.c.) was effective for 15 days, demonstrating a slower trend in tumour growth compared with control. However, after day 15, tumours had increased growth rates and mice were killed at day 36. Everolimus (2.5 mg kg−1 every 2 weeks p.o.) was effective in reducing tumour volume for the first 30 days of the experiment, after that tumour growth was rapid and exponential. Dual inhibition of bicalutamide plus everolimus was more effective than either bicalutamide or everolimus alone at day 35 (3 after 43 days at termination of the experiment (Control tumours grew rapidly and mice were killed 27 days after the start of the treatment . VN/124-t day 35 . Howevert day 35 . Inhibitperiment . All treP<0.003) and also bicalutamide (P<0.0001). Therefore, the addition of VN/124-1 to everolimus therapy proved to be superior to everolimus therapy alone in the HP-LNCaP model. There was no increase in tumour growth after day 25 in the VN/124-1 plus everolimus-treated group  was compared between the controls and each of the five treatment groups. The growth curves were estimated over 29 days, but there were no differences found in growth rate among these groups. However, pairwise comparisons of mean tumour volume on days 29 and 43 demonstrated a statistically significant difference between VN/124-1 plus everolimus and everolimus , in the combination of VN/124-1 plus everolimus group, was also statistically significantly lower than all other treatment groups.Serum and tumour PSA levels were also analysed. As expected, PSA concentrations in serum were significantly lower than in the tumour tissue. Mean PSA in serum was 8.36 ng mlin vitro findings , p-MAPK (3-fold), p-mTOR (2.2-fold), and p-P70S6K (4-fold) . Interesin vitro in HP-LNCaP cells and in vivo in the LNCaP tumours with anti-androgen treatment. As shown in P=0.0001) or everolimus (P=0.04). Additionally, in the HP-LNCaP xenograft model (−1 q.w. p.o.) plus VN/124-1 (100 mg kg−1 q.d. s.c.) significantly (P=0.03) reduced tumour volumes and growth rates compared with single-agent everolimus. Furthermore, growth trends indicate that VN/124-1 plus everolimus was more effective than bicalutamide plus everolimus. Although PSA levels in tumour tissue were higher than in serum, nevertheless, PSA levels in serum and tissues were reduced only by VN/124-1 plus everolimus, consistent with tumour growth measurements, and indicate the greater anti-tumour efficacy of VN/124-1 than bicalutamide. It is of note that, although serum levels were reduced to undetectable levels, tissue levels were still measurable, consistent with incomplete tumour suppression.Lack of effective treatment of CRPC represents a significant problem in current management of PCa. Despite initial responses to hormonal therapy, nearly all advanced PCa patients eventually fail treatment, as tumours become resistant. This investigation was designed to understand the molecular mechanisms that enable androgen-dependent PCa cells to progress to CRPC. We also sought to determine whether blocking the resistance pathway would restore sensitivity to standard anti-androgens and/or to our novel agent VN/124-1. Improved understanding of the interaction between the AR and growth factor signalling could yield a better strategy for treating disease progression, which may delay, re-sensitise, or prevent CRPC. The data suggest that increased sensitivity to androgens and a compensatory cross-talk mechanism between the AR and mTOR could have a role in the progression to androgen independence. In the two models used here, resistance to androgen deprivation was developed ft model , dual inVN/124-1 inhibits androgen synthesis, as well as binds to the AR and causes its downregulation (In vivo findings also demonstrate that inhibition of AR via bicalutamide or VN/124-1 increases the expression and activation of several growth factor signalling proteins. Compensatory cross-talk was evident by changes in proteins in AR, the MAPK, and P13K/Akt/mTOR pathways, Thus, inhibition of mTOR increased AR protein expression in vitro as well as in vivo (in vivo. We found that serum PSA levels, but not tissue levels, were suppressed by bicalutamide and VN/124-1, although tumour growth was not inhibited. This suggests that tissue PSA may reflect tumour growth more accurately, and that only a portion of the PSA produced enters circulation. It is also possible that AR-regulated PSA is still responsive to anti-androgens, whereas tumour growth is independent of androgens and is responsive to other signalling pathways such as mTOR.Our results indicate that blockade of AR with anti-androgens and of mTOR with everolimus improved sensitivity to these agents. Although others (In summary, our studies show that interaction between the AR and mTOR pathways appears to be involved in tumour progression in the xenograft models. There is an increase in signal transduction protein expression as tumours progress on anti-androgen therapy and in control tumours that are actively growing. Bicalutamide and everolimus treatment reduced tumour growth to some extent in comparison with that of control tumours in the resistant models. However, by combining mTOR inhibition with an anti-androgen to block both the pathways from the beginning, tumour growth was statistically significantly reduced compared with the effect of these drugs alone in the LNCaP and HP-LNCaP xenografts. Thus, there may be value in using an mTOR inhibitor in combination with an AR downregulator such as VN/124-1. VN/124-1 in combination with an mTOR inhibitor may reduce AR levels and thereby delay transition to growth factor receptor signalling and hormone resistance. Although everolimus in combination with bicalutamide has not been found to be effective in PCa patients (


The monoclonal antibody A33 recognises a tumour-associated antigen on human colorectal carcinoma, and has undergone preliminary evaluation in the clinic where selective localisation to hepatic metastases has been demonstrated . A33 and an A33 tri-fab fragment (TFM) were labelled with 90Y via a stable macrocyclic ligand for biodistribution and therapy studies in nude mice bearing SW1222 colon carcinoma xenografts. Biodistribution studies demonstrated tumour localisation for both A33 IgG and TFM with low bone, liver and kidney levels. Clearance of TFM from the blood was much faster than IgG and this led to lower tumour accumulation for TFM but superior tumour-blood ratios. The maximum per cent injected dose per g localised to tumour was 35.9% +/- 5.3% for A33 IgG and 12.9% +/- 4.6% for A33 TFM with tumour-blood ratios at 48 h after administration of 5.6 +/- 1.8 and 29.2 +/- 9.8 respectively. Autoradiography studies with 125I-labelled A33 IgG and TFM demonstrated a homogeneous distribution within tumour tissue which was not observed with other anti-colorectal tumour antibodies. TFM penetrated into the tumour tissue more rapidly than IgG. In therapy studies, a single dose of 90Y-A33 IgG (250 microCi per mouse) or 90Y-A33 TFM (300 microCi per mouse) led to complete regression of 2-week-old tumour xenografts with long-term tumour-free survivors. A transient drop in white blood cell count was observed with both IgG and TFM but was significantly more pronounced with IgG. The cell count fell to 8.4% of control for IgG, whereas with TFM cell counts fell to 51% of control before recovery. These results indicate that the more rapid blood clearance of 90Y-TFM confers reduced toxicity compared with 90Y-IgG although similar therapeutic effects are achieved. When the dose of 90Y-IgG was adjusted to give the same dose to tumour achieved with 300 microCi 90Y-TFM, a lesser therapeutic effect was observed. This may be owing to more rapid tumour penetration achieved with TFM. Both A33 IgG and TFM demonstrated potent anti-tumour effects against human tumour xenografts in this mouse model system. The stability of these 90Y-labelled conjugates and their effective tumour penetration are promising for the development of humanised reagents for clinical studies.


One of the main obstacles encountered when trying to culture human seminoma (SE) cells in vitro is massive degeneration of the tumour cells. We investigated whether dissociation of tumour tissue, to obtain single-cell suspensions for in vitro culture, results in the onset of apoptosis. Using morphological analysis and in situ end labelling, less than 4% of apoptotic tumour cells were detected in intact tissue from 11 out of 14 SEs. In these 11 tumours, apoptosis-specific DNA ladders, indicative of internucleosomal double-strand DNA cleavage, were not detected on electrophoresis gels. In contrast, three SEs with over 12% of apoptotic tumour cells in the intact tissue and all analysed (pure) SE cell suspensions, obtained after mechanical dissociation of intact tumour tissue, showed DNA ladders. Flow cytometric analysis of end labelled SE suspensions showed DNA breaks in up to 85% of the tumour cells. As indicated by cell morphology and DNA degradation, SE cells appear to rapidly enter the apoptotic pathway upon mechanical disruption of their microenvironment. No expression of p53 and of the apoptosis-inhibitor bcl-2 was detectable in intact SE tissue or cell suspensions. Our data suggest that abrogation of apoptosis might be crucial to succeed in culturing human SE cells in vitro.


Serum levels of cell-free interleukin-2 receptors were elevated above normal in mice bearing the IL-2R positive T-cell lymphoma Eb or its highly metastatic variant ESb. Although ESb cells expressed less IL-2R molecules than Eb cells on their cell surface, serum receptor levels were raised more quickly in ESb than in Eb tumour bearing animals. Elevated IL-2R serum levels were a sensitive tumour marker in animals bearing the aggressive variant ESb but not in animals bearing the low metastatic line Eb. Peritoneal ascites tumour-bearing animals had higher serum IL-2R levels than corresponding animals with subcutaneously growing tumours. Thus, serum IL-2R levels in tumour-bearing animals were dependent on the tumour line and influenced by the site and mode of tumour growth.


Immunofluorescence studies of sera from mice with induced enhancement of tumour growth demonstrated that these sera contained factors ("interfering factors") which in an apparently competitive manner interfered with the subsequent binding of specific antibodies to antigenic sites on the tumour-cell membrane. The factors were tumour-specific but lacked some of the immunoglobulin determinants. They could not be detected by polyvalent FITC-antimouse gamma-globulin. Interfering factors did not seem to be related to IgA or IgE. They were demonstrable in sera from tumour-free animals without growing tumours, thus differing from the tumour-specific "blocking factors".


ADEPT is an antibody-based targeting strategy for the treatment of cancer. We have developed two new prodrugs, 4--phenoxycarbonyl-L- glutamic acid (PGP) and (S)-2-- phenoxycarbonyl]amino]-4-(5-tetrazoyl)butyric acid (PTP), which are cleaved by the bacterial enzyme CPG2 to release the 4- phenol drug. In vitro, both prodrugs are approximately 100- to 200-fold less potent than the parent drug (1 h IC50 = 1.4 microM) in LoVo colorectal tumour cells. These prodrugs have been evaluated for utility in ADEPT when used in combination with a conjugate of CPG2 and the F(ab')2 fragment of the anti-CEA monoclonal antibody, A5B7. The conjugate was shown to localise specifically to established LoVo tumour xenografts growing in nude mice and optimal tumour-normal tissue ratios were achieved after 72 h. Administration of either prodrug, at doses which cause 6-8% body weight loss, 72 h after administration of the A5B7-CPG2 conjugate to the LoVo tumour-bearing mice resulted in tumour regressions and growth delays of 14-28 days. The PTP prodrug in combination with a high dose of conjugate (10 mg kg-1) gave the best anti-tumour activity despite being a 10-fold worse substrate for CPG2 than PGP. Prodrug alone, active drug alone or prodrug in combination with a non-specific conjugate had minimal anti-tumour activity in this tumour model.


A mathematical model was developed to improve understanding of the biodistribution and microscopic profiles of drugs and prodrugs in a system using enzyme-conjugated antibodies as part of a two-step method for cancer treatment. The use of monoclonal antibodies alone may lead to heterogeneous uptake within the tumour tissue; the use of a second, low molecular weight agent may provide greater penetration into tumour tissue. This mathematical model was used to describe concentration profiles surrounding individual blood vessels within a tumour. From these profiles the area under the curve and specificity ratios were determined. By integrating these results spatially, average tissue concentrations were determined and compared with experimental results from three different systems in the literature; two using murine antibodies and one using humanised fusion proteins. The maximum enzyme conversion rate (Vmax) and the residual antibody concentration in the plasma and normal tissue were seen to be key determinants of drug concentration and drug-prodrug ratios in the tumour and other organs. Thus, longer time delays between the two injections, clearing the antibody from the blood stream and the use of 'weaker' enzymes (lower Vmax) will be important factors in improving this prodrug approach. Of these, the model found the effective clearance of the antibody outside of the tumour to be the most effective. The use of enzyme-conjugated antibodies may offer the following advantages over the bifunctional antibody-hapten system: (i) more uniform distribution of the active agent; (ii) higher concentrations possible for the active agent; and (iii) greater specificity (therapeutic index).


The proliferation characteristics of vascular endothelium have been studied in 131 individual experimental tumours, representing 18 transplanted tumour lines. The labelling index (LI) is high in most tumours, with a mean value of 0.9%, regardless of the growth rate of the tumours, or whether different tumour types are considered or individual tumours from within one line are studied in detail. A similar high LI value has been found by others for a human tumour. These high LI values may even underestimate the proliferation in new capillary buds. The high proliferative index of tumour endothelium is in marked contrast with the previously reported low 3HTdR uptake into normal tissue blood vessels. It seems likely that it is the type of new vessels formed that will influence tumour growth rates more than the simple rate of endothelial-cell proliferation. The large difference between the proliferation characteristics of tumour endothelium and normal tissue endothelium, recently identified as a possible approach for tumour therapy, has now been confirmed for a range of animal tumours and a human tumour.


Lipid metabolism has been considered recently as a novel target for cancer therapy. In this field, lithium gamma-linolenate (LiGLA) is a promising experimental compound for use in the treatment of human tumours. In vivo and in vitro studies allowed us to assess the metabolism of radiolabelled LiGLA by tumour tissue and different organs of the host. In vitro studies demonstrated that human pancreatic (AsPC-1), prostatic (PC-3) and mammary carcinoma (ZR-75-1) cells were capable of elongating GLA from LiGLA to dihomo-gamma-linolenic acid (DGLA) and further desaturating it to arachidonic acid (AA). AsPC-1 cells showed the lowest delta5-desaturase activity on DGLA. In the in vivo studies, nude mice bearing the human carcinomas were given Li[1-(14)C]GLA (2.5 mg kg(-1)) by intravenous injection for 30 min. Mice were either sacrificed after infusion or left for up to 96 h recovery before sacrifice. In general, the organs showed a maximum uptake of radioactivity 30 min after the infusion started (t = 0). Thereafter, in major organs the percentage of injected radioactivity per g of tissue declined below 1% 96 h after infusion. In kidney, brain, testes/ovaries and all three tumour tissues, labelling remained constant throughout the experiment. The ratio of radioactivity in liver to tumour tissues ranged between 16- and 24-fold at t = 0 and between 3.1- and 3.7-fold at 96 h. All tissues showed a progressive increase in the proportion of radioactivity associated with AA with a concomitant decrease in radiolabelled GLA as the time after infusion increased. DGLA declined rapidly in liver and plasma, but at a much slower rate in brain and malignant tissue. Seventy-two hours after the infusion, GLA was only detected in plasma and tumour tissue. The sum of GLA + DGLA varied among tumour tissues, but it remained 2-4 times higher than in liver and plasma. In brain, DGLA is the major contributor to the sum of these fatty acids. Data showed that cytotoxic GLA and DGLA, the latter provided either by the host or by endogenous synthesis, remained in human tumours for at least 4 days.


When administered to mice bearing the MAC16 tumour, CV-6504 rapidly disappeared from the plasma and tissues and there was an accumulation of the sulphate and glucuronide metabolites. After 24 h, the concentration of free CV-6504 in the tumour (3.3 microM) was higher than that in the liver (0.24 microM) and equal to the IC50 value for the inhibition of the growth of MAC16 cells in vitro (3 microM). The concentration of glucuronide and sulphate metabolites in both tumour and liver decreased with time. Both the MAC16 tumour and the liver possessed similar beta-glucuronidase activity, which could account for the accumulation of free CV-6504. Although the sulphate and glucuronide conjugates of CV-6504 were ineffective inhibitors of the growth of MAC13 cells in vitro at concentrations up to 100 microM, in vivo at a concentration of 50 mg kg-1 day-1 the conjugates produced a similar anti-tumour effect to CV-6504 at a concentration of 5 mg kg-1 day-1. The MAC13 tumour possessed both beta-glucuronidase and sulphatase activity capable of converting the sulphate and glucuronide conjugates to free CV-6504. Using MAC13 cells ex vivo, CV-6504 inhibited conversion of arachidonic acid to 5-, 12- and 15-hydroxyeicosatetraenoic acids (HETE). The percentage reduction in formation of 12- and 15-HETE exceeded that of 5-HETE. Inhibition of HETE formation may be responsible for the anti-tumour activity of CV-6504.


The analyses were carried out with the aid of ion-exchange chromatography. The molar proportions of 5 bases detected in acid hydrolysates of tumour tRNA (N


Tumour-associated monoclonal antibodies  radiolabelled with iodine-131 were given intracavitary  to patients with malignant effusions. Ten out of 13 effusions  responded completely with no fluid reaccumulation between 3 and 18 months. No clinical or other toxicity was observed. This new method of treatment for recurrent malignant effusions is non-toxic and effective resulting in improved quality of life, and, in some cases, prolongation of survival.


A comparison has been made of the effects of the 2-nitroimidazole Ro-07-0582 on tumour growth delay after irradiation and tumour cell survival in vitro after irradiation in vivo. This compound has previously been shown to be a specific sensitizer of hypoxic cells. A dose of 1 mg/g body weight gave an enhancement ratio of 2-2 for both growth delay and cell survival in a system where high pressure oxygen has been shown to have no effect. However, while the hypoxic fraction in the tumour was estimated to be less then 10% from the growth delay curves, the survival curves gave a value in excess of 50%. This discrepancy probably reflects differences in the response of cells left in situ or removed and assayed in vitro.


Different factors contribute to the onset of labor at term. In animal models onset of labor is characterized by an inflammatory response. The role of intrauterine inflammation, although implicated in preterm birth, is not yet established in human term labor. We hypothesized that intrauterine inflammation at term is associated with spontaneous onset of labor.In two large urban hospitals in the Netherlands, a cross-sectional study of spontaneous onset term vaginal deliveries and elective caesarean sections (CS), without signs of labor, was carried out. Placentas and amniotic fluid samples were collected during labor and/or at delivery. Histological signs of placenta inflammation were determined. Amniotic fluid proinflammatory cytokine concentrations were measured using ELISA. A total of 375 women were included. In term vaginal deliveries, more signs of intrauterine inflammation were found than in elective CS: the prevalence of chorioamnionitis was higher  and amniotic fluid concentration of IL-6 was higher . Similar results were obtained for IL-8  and percentage of detectable TNF-α .This large cross-sectional study shows that spontaneous term delivery is characterized by histopathological signs of placenta inflammation and increased amniotic fluid proinflammatory cytokines. Normal human delivery is initiated by the spontaneous onset of labor at term.The clinical relevance of sterile inflammation in normal delivery is intriguing. Inflammation may provide a danger signal by the innate immune system, resulting in the onset of labor.A cross-sectional study was conducted in one secondary and one tertiary hospital in Utrecht, The Netherlands, between January 2006 and November 2007. Women delivering vaginally at term after an uncomplicated pregnancy and with spontaneous onset of labor were recruited. In addition, women who delivered by elective CS were recruited. Elective CS was defined as a planned CS, performed in the absence of signs of labor or rupture of membranes. Women were excluded if they did not comprehend the Dutch language, if the gestational age was below 37 weeks, or if the infant had major congenital organ abnormalities . Recruitment was performed by one of the researchers. Shortly before or after delivery, parents were informed about the study. Collected samples of the placenta and amniotic fluid were only analysed when parents consented. The study was approved by the ethical review boards of both institutions and all parents provided written informed consent for study participation.Clinical characteristics of the mother  and her child  were collected. Gestational age was determined with the use of clinical history and the results of the earliest ultrasound examination. Secondary CS was defined as a CS performed after the onset of labor. Maternal fever during delivery was defined as a temperature of 38.0°C or higher, as measured during labor and delivery. Newborn infection was defined as a strongly suspected or proven early onset neonatal sepsis, within 48 hours after birth.Placentas were stored at +4°C and processed within 72 hours. In case of vaginal delivery, amniotic fluid was collected vaginally, in a non-sterile manner, at the moment of artificial rupture of membranes or after spontaneous rupture of membranes. When spontaneous rupture of membranes occurred outside the hospital, collection of amniotic fluid was usually performed immediately after birth, because at that moment a larger volume of amniotic fluid passes the vagina. During elective CS, amniotic fluid was collected with a syringe directly after incision of the membranes. Amniotic fluid samples were stored at +4°C and processed within 72 hours.Placenta examination was performed by an experienced pathologist (PN).After macroscopic assessment, the samples were purified by filtration  and centrifugation  to remove debris and blood clots. The supernatant was filtered through a 0.2 µL sterilization filter  to remove leukocytes and other contaminants. The sterile, acellular fraction of amniotic fluid was stored at −80°C until further analysis. Enzyme linked immunosorbent assays (ELISA) were used to determine interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) concentrations . The lower limits of detection for IL-6, IL-8 and TNF-α were 0.006 ng/mL, 0.01 ng/mL and 0.014 ng/mL, respectively. IL-6 and IL-8 concentrations were never undetectable. Amniotic fluid cellular composition was not systematically determined.Several pilot experiments were conducted to validate the methods of amniotic fluid analysis.(a) The influence of storage at +4°C during 72 hours was studied in paired fresh and stored samples. (b) To minimize the effect of aspecific binding proteins, spiking of diluted amniotic fluid with synthetic cytokines was performed to find the optimal dilution for ELISA. (c) Intra-assay and interassay reproducibility were determined by comparing the results of duplo amniotic cytokine measurements within and between ELISA assays. (d) The effect of different intervals from labor until amniotic fluid sampling or from sampling until delivery between individuals was studied by collection and analysis of serial samples. (e) The influence of delay of amniotic fluid sampling after spontaneous rupture of membranes was analysed by comparing deliveries with different delay intervals. (f) To study whether transvaginal collection of amniotic fluid affects cytokine concentrations, paired samples were collected transvaginally and using an intrauterine catheter simultaneously.2 or Fisher's Exact test. The geometric mean amniotic fluid IL-6 and IL-8 concentrations were compared using the Student's T test. The proportion of detectable TNF-α was compared using the Pearson's χ2 test. Unadjusted odds ratios (OR) of chorioamnionitis, funisitis, villitis, amniotic fluid IL-6, IL-8 and detectable TNF-α for term vaginal delivery were calculated. With multivariable logistic regression analysis, OR were adjusted for potential confounders, i.e. maternal age, gestational age, parity, meconium-staining. These potential confounders were selected for their biological plausibility and for their correlation to both intrauterine inflammation and term vaginal delivery. Collinearity between amniotic fluid cytokine concentrations was studied by calculating correlation coefficients. All data were analysed in the Statistical Package for Social Sciences (SPSS) version 15.0.The association between intrauterine inflammation and term vaginal delivery was studied. This cross-sectional study was part of a larger birth cohort study, for which a sample size of 500 participants was calculated; a sample size calculation for this study was not performed. Logarithmic transformation was used for amniotic fluid IL-6 and IL-8 concentrations, to provide a normal distribution. TNF-α was dichotomized into detectable and undetectable, because a large proportion of the samples contained undetectable levels of TNF-α. The frequency of chorioamnionitis, funisitis and villitis was compared between term vaginal deliveries and elective CS using the Pearson's χDuring the study period, 1190 term vaginal deliveries occurred.and 154 elective CS were performed. Informed consent was obtained from 375 mothers. ClinicalThe women with spontaneous onset of labor, were younger, had a higher gestational age and a higher proportion of meconium stained amniotic fluid, as compared to the women in the elective CS group . MaternaPlacenta histology was established in 317 samples. The remaining 58 placentas were not collected for non-specific reasons. Chorioamnionitis was present in 50 of 271 placentas (18%) in the group of women with spontaneous onset of labor and in 2 of 46 placentas (4%) in the elective CS group  6.6–21.6, p = 0.02) . FunisitAmniotic fluid cytokines were determined in 373 samples. In the group of women with spontaneous onset of labor, amniotic fluid IL-6 concentrations were higher than in elective CS , suggesting that these cytokines play a role in the same inflammatory event, but do not have an identical expression pattern. Chorioamnionitis was only weakly correlated with amniotic fluid IL-6, IL-8 and TNF-α .(a) Amniotic fluid cytokine concentrations measured in fresh samples were similar after storage. (b) Spiking of amniotic fluid with synthetic cytokines showed good reliability of dilution of amniotic fluid . (c) Amniotic fluid cytokine measurements had a high intra-assay and interassay reproducibility . (d) We did not find an association between duration of labor until sampling, or duration from sampling until delivery and cytokine concentrations in amniotic fluid . (e) Delay of sampling after spontaneous rupture of membranes did not result in different amniotic fluid cytokine concentrations . (f) AmnIn this cross-sectional study of 375 term vaginal deliveries and elective CS, we found that signs of intrauterine inflammation are associated with spontaneous onset of labor. Histological signs of chorioamnionitis were found in 18% of placentas of term vaginal deliveries, whereas acute inflammation was virtually absent in elective CS. Amniotic fluid IL-6 and IL-8 concentrations and the proportion of deliveries with detectable amniotic fluid TNF-α were approximately tenfold higher in term vaginal deliveries than in elective CS.Intrauterine inflammation has been shown to induce preterm labor in a primate model. Intra-amniotic injection of TNF-α and IL-1β rapidly induced onset of labor.Ureaplasma Urealyticum, have been associated with the onset of labor.The origin of intrauterine inflammation at term is uncertain but might be infectious. An estimate of 18% of women will have microbial invasion of the amniotic cavity at term, increasing to 30% when there is prolonged rupture of the membranes.The major strengths of this study are that it examined evidence of inflammation in different intrauterine compartments and the large sample size. Amniotic fluid samples were collected vaginally during active labor. Other studies have collected amniotic fluid by amniocentesis or in pregnant women presenting with preterm contractions.Some possible limitations should also be discussed. The cross-sectional design of data collection does not allow definite conclusions on causality. The inflammation might also be the result of birth process, including maternal stress by labor activity or fetal stress by exposure to contractions during labor and passage through the birth canal.The results of this study may have clinical implications. Better insight in the physiological role of intrauterine inflammation may eventually result in strategies to intervene in normal labor and delivery, including induction of labor in postterm pregnancies.In conclusion, human term spontaneous labor and delivery are associated with a high level of intrauterine inflammation, and intrauterine inflammation is a consistent finding in all intrauterine compartments. This large cross-sectional study shows that normal labor at term is characterized by chorioamnionitis in a considerable number of cases and high amniotic fluid concentration of proinflammatory cytokines in all cases, providing insight in the pivotal role of intrauterine inflammation in normal parturition.


The effect of 16,16-dimethyl-PGE2-methyl ester (di-M-PGE2), a long-acting synthetic analogue of prostaglandin E2, on the replication of Friend erythroleukaemia cells (FLC) in vivo has been studied. Pre-treatment in vitro of both undifferentiated and differentiated FLC with di-M-PGE2 (1 microgram/ml) did not alter rates of tumour appearance or growth, but increased the median survival of DBA/2J mice. Systemic administration of di-M-PGE2 (10 microgram/mouse/day) was not toxic to the mice, but significantly inhibited tumour growth and increased median survival in mice injected s.c. with undifferentiated FLC. These effects of di-M-PGE2 were much more pronounced in mice receiving differentiated (DMSO-treated) FLC. In this latter group, the appearance of tumour was also significantly delayed by di-M-PGE2. The different effects of di-M-PGE2 treatment on tumours derived from undifferentiated and differentiated cells suggest that the analogue is acting directly on tumour-cell replication rather than on factors related to the host response.


Hyaluronan (HA), an extracellular high-molecular-mass polysaccharide, is supposed to be involved in the growth and progression of malignant tumours. We studied the cellular expression of HA in 215 operated stage I–IV gastric cancer patients using a specific biotinylated probe. Most (93%) of the tumours showed HA staining in their parenchyma, whereas the stroma inside and around the tumour was stained in every case. When HA expression was compared with the clinical and histological features of the tumours, a strong staining intensity in the tumour parenchyma was found to be associated with deep tumour invasion (pT3 or 4) and with mixed type of Laurén. A high proportion of HA-positive cells of all neoplastic cells was significantly associated with deep tumour invasion, nodal metastasis, positive lymphatic invasion, poor differentiation grade, as well as with inferior prognosis in univariate survival analysis. However, in multivariate analysis, only pT, pN, and vascular and lymphatic invasion emerged as independent predictors of survival in gastric cancer. The results indicate that ectopic HA expression is a frequent feature of gastric adenocarcinoma, and is associated with tumour progression and poor survival rate. © 1999 Cancer Research Campaign


Compared to the parental cells, INT.ACP/PTX cells exhibited a high level of Pgp expression, resulting in a reduced in vitro sensitivity to both PTX and IDN 5390. The INT.ACP/PTX tumour xenograft was still resistant to PTX, but responsive to IDN 5390, when delivered per os, by a daily prolonged schedule. A direct effect on tumour cells, allowed by the high tolerability of the compound in mouse, cannot be excluded in vivo. Immunohistochemical analysis indicated a significant reduction of microvessel density in IDN 5390-treated tumours, lasting till 7 days after the last drug administration. Thus, a prolonged inhibitory effect on tumour angiogenesis is consistent with the persistent growth control of INT.ACP/PTX tumour achieved by IDN 5390. On the contrary, the low tolerability and the limited oral availability of conventional taxanes do not allow an easy feasibility of such treatment regimen. Thus, the tolerability profile of IDN 5390 in preclinical systems and its efficacy in PTX-resistant tumours support the therapeutic interest for its clinical development, with particular attention to oral daily prolonged schedules.IDN 5390 is a novel C-seco taxane analogue selected for preclinical development on the basis of its antimotility activity on endothelial cells, antitumour efficacy in a large panel of human tumour xenografts and high tolerability in mouse. On the basis of oral availability, IDN 5390 is suitable for protracted administration schedules. Such a treatment schedule has been reported as the most appropriate to exploit the antiangiogenic effects of cytotoxic drugs. An ability to downregulate angiogenesis-related growth factors in tumour cells has been described for IDN 5390. The aim of the study was to investigate the antitumour and antiangiogenic potential of oral IDN 5390 on a human ovarian carcinoma xenograft, the INT.ACP/PTX, resistant to paclitaxel (PTX). Such tumour line was derived  In vivo studies in human tumour xenografts have indicated a unique profile of tolerability and antitumour activity for IDN 5390 when administered by a daily schedule for a prolonged time  after treatment with PTX. The mechanisms of resistance of these tumour cells and the in vivo antiangiogenic effect of the drug were investigated. The results are consistent with a contribution of antiangiogenic effects in the antitumour efficacy of the taxane analogue.The aim of the study was to further investigate the mechanisms responsible for the antitumour activity of IDN 5390 in PTX-resistant human tumour xenografts and, in particular, in the ovarian carcinoma INT.ACP/PTX. Such tumour line was derived N-Boc-β-isobutylisoserinyl)-10β-dehydro-C-seco-10-deacetylbaccatin III) and PTX, used as reference drug, were provided by Indena S. p.a.   at 2 mg ml−1 and diluted in culture medium . For in vivo studies, PTX was dissolved by adding absolute ethanol and Cremophor ELP   supplemented with 10% foetal calf serum  in 5% CO50) was derived by dose/effect plots. Resistant Index (RI) was assessed as the ratio between the drug IC50 in each cell subline and in the parental A2780 cell line.Tumour cell sensitivity to IDN 5390 and PTX was evaluated by cell growth inhibition assay. Cells were seeded in six-well plates in duplicate and after 24 h exposed to the solvent or to the drugs at different concentrations. After 72 h, cells were trypsinised and counted by a Coulter Counter . The concentration able to inhibit cell proliferation by 50% (IC6) were trypsinised and washed in PBA  and 1% bovine serum albumin (BSA)). Cells were then incubated in 100 μl of PBA containing 10 μl of gp170-FITC antibody . After washing in PBS, expression of the protein was assessed as fluorescence intensity using a FACScan .For the MDR phenotype characterisation, the Pgp expression was determined by immunofluorescence . Cells (Bcl-2 expression was examined in whole-cell extracts prepared as previously reported in All the experiments were carried out using adult (8–10 weeks old) female athymic CD-1 nude mice . Mice were maintained in laminar flow rooms at constant temperature and humidity, with free access to sterilised food and water. Experimental protocols were approved by the Ethic Committee for Animal Experimentation of our Institute , according to the United Kingdom Coordinating Committee on Cancer Research Guidelines , and in such conditions tumour weight inhibition (TWI) induced by the optimal PTX regimen was lower than 50 vs a 94% inhibition in the A2780/DDP tumour line =d2 × D/2, where d and D are the shortest and the longest diameter, respectively. Mice bearing tumours of 250–300 mg were treated with IDN 5390 and PTX, according to different treatment routes  and schedules . The first and the last day of treatment were the same for the two schedules. At 1 and 7 days after treatment end, three mice/group were killed by cervical dislocation. Tumours were excised, weighed, fixed in zinc fixative and stained with standard haematoxylin—eosin in order to study the overall tissue morphology. Angiogenesis was assessed by immunohistochemistry (IHC), staining blood vessels with a rat anti-mouse CD31/PECAM-1 monoclonal antibody , as previously described  selected for high vascularisation (hot spot areas). Microvessel density was expressed as mean number±s.d. Percentage MV inhibition (MVI%) in drug-treated vs control mice was calculated as MVI%=100–(mean MVD in treated/mean MVD in control tumours × 100). Neither vessel lumen nor red blood cells were used to define a microvessel. Scoring of histological tumour sections was performed by two independent observers, without knowledge of the experimental group, with an interobserver reproducibility >95%.For experimental purposes, tumour fragments were s.c. implanted in the right flank of mice. Tumour growth was followed by biweekly measurements of tumour diameters with a Vernier caliper. Tumour weight (TW) was calculated according to the formula: TW (mg)=tumour volume  expressed as TWI%=100–(mean TW treated/mean TW control × 100).vs control mice by the unpaired Student's t-test (two-tailed).For statistical analysis, TW and MV number were compared in treated Pgp overexpression is a frequent mechanism of the cellular resistance to hydrophobic drugs, including PTX  resulted in a complete reversal of resistance, with IC50 values similar to those in the A2780/DDP cell line, that is, 17.5±1.2 and 57±4 nM for PTX and IDN 5390, respectively (not shown).The antiproliferative effects of PTX and IDN 5390 after 72 h of treatment indicated an inverse correlation between Pgp expression and cell sensitivity to the taxanes , or at its best regimen (P<0.05).We have already reported that the INT. ACP/PTX tumour xenograft is resistant to PTX (less than 50% TWI), but highly sensitive (87% TWI) to IDN 5390, 120 mg kgvs control tumours . Paclitaxel was administered daily by the s.c. route, due to its very poor oral bioavailability (P<0.05). Paclitaxel administered i.v. at its best regimen for antitumour activity (high-dose/intermittent schedule) had limited effect on tumour angiogenesis.A prolonged treatment schedule, which resulted to be the optimal one for the antitumour efficacy of IDN 5390, has been reported as the most suitable for exploiting the antiangiogenic effect of cytotoxic drugs. Thus, the effect on angiogenesis of the novel taxane was assessed in INT.ACP/PTX tumour xenografts , whereas a superior antitumour effect (87% TWI) was achieved by the same dose twice a day for 2 weeks  after oral administration of a single dose (120 mg kg−1) are high enough to support our interpretation , but a lower effect on MVD. Inhibition of angiogenesis in the INT.ACP/PTX tumour xenograft was achieved also by PTX delivered by the daily s.c. treatment. The reduction on MVD was similar to that achieved by the analogue, but PTX-induced effect was less persistent. No antitumour efficacy was achieved by PTX delivered by these conditions. In spite of a similar ability to inhibit tumour angiogenesis, the limited exposure of the tumour cells to PTX, due to the low drug tolerability in vivo, may account for the lack of efficacy of PTX. In addition, the higher level of resistance  may be a further drawback of PTX. Moreover, considering the very poor oral bioavailability of PTX  and still present 1 week after (26% MVI). Such finding was in keeping with other preclinical in vivo and the oral bioavailability allow exploiting the therapeutic potential of high doses of the drug  throughout a continuous and prolonged period of treatment (favouring effect on angiogenesis inhibition). Although the therapeutic advantages of IDN 5390 over PTX could be achieved only at relatively high dose levels, the favourable pharmacological profile makes IDN 5390 a promising candidate for clinical development.In conclusion, the relevant antitumour activity of the novel taxane IDN 5390 against a PTX-resistant tumour xenograft is likely to be the result of multiple actions, favoured by the peculiar pharmacological properties of the compound. Indeed, the good tolerability