The N-Nitrosomethylurea breast cancer model induced in rats is used for the study of carcinogenesis in mammary cancer, prostate, pancreas, etc. This model is very similar to human neoplastic disease.The present experimental study was designed to assess whether metoclopramide administration has any effect on development of MNU-induced tumours, and evaluate the treatment of goserelin acetate on PRL, TNF alpha and NO expression. NMU was administered to female Wistar rats on 2 occasions (5 mg/100 g body w/rat). PRL and TNF alpha were performed by immune-assay. Nitric Oxide by semi automated-assay and ploidy analyses by flow cytometry.The administration of metoclopramide made the induction time shorter and increased the incidence and average of tumours per rat. Tumours development was inhibited by a goserelin chronic administration. The ploidy of adenocarcinoma was polyploid-aneuploid type (average S = 60%). It was higher basal PRL plasma levels in rats with NMU induced tumours than in basal controls without tumour (p < 0.001). The goserelin "in bolus" administration showed maximal inhibition of plasma PRL at 90 min. Plasmatic TNF alpha expression was inhibited at 60 min and also remained inhibited in tissue homogenate post chronic treatment (P < 0.0125). Plasmatic NO expression is higher in rats with induced tumours than healthy controls (P < 0.001). In tissue homogenate NO values were inhibited at 90 min (P < 0.01), as well during chronically goserelin treatment (P < 0.005).The increase of blood PRL levels in NMU-induced rats may be an indicator of a poor prognosis of mammary cancer evolution. The metoclopramide administration accelerates tumour growth. However goserelin administration achieves regression in tumour development associated to inhibition PRL, TNF alpha and NO expression. N-nitroso-N-methylurea (NMU) is an effective chemical carcinogen for the induction of mammary carcinoma in rats and a very good model for human mammary carcinomas -6. Prola14H22ClN3O2), their IUPAC name: 4-amino-5-chloro-N-(2-diethylamino)ethyl-2-methoxybenzamide is a potent dopamine receptor antagonist used for its antiemetic effect and by inhibiting the action of PRL inhibiting hormone and has sometimes been used to stimulate lactation. It thus may induce hyperprolactinemia in rats.Metoclopramide  is known to suppress ovarian function and plasma prolactin levels and its antitumour activity has been evaluated extensively .In chemical carcinogenesis, internal cytokines, such as interleukin (IL) 1α and tumour necrosis alpha (TNFα) contribute to tumour progression. However it has been suggested that TNF on tumour vasculature has a direct anti-tumour mechanism -14. IndeInducible nitric oxide synthase (iNOS) and nitric oxide (NO) are implicated in tumour pathology and, it is well known that TNFα induces iNOS and NO release. While low concentrations of NO alter transcription factor and act as an antioxidant factor, high concentrations of NO can damage DNA and proteins. Moreover NO-induced alteration in cellular function can lead to cell death or stimulation of growth, and stimulate tumour growth by promoting angiogenic and invasive capacities -19.The aim of the study was to assess the effect of metoclopramide on tumour development of NMU-induced tumours in Wistar rats, and at the same time to study acute and chronic administration of  goserelin on PRL, TNFα and NO expression in this experimental model.Wistar rats were used for all the experiments and were obtained at 45 days of age. Animals were housed 2/cage in a room illuminated for 12 h each day at a temperature of 25°C. The animals had free access to Purina laboratory chow and drinking water. All the experiments in this study were performed in compliance with the Royal Decree 223/1988 of March (BOE 8 18) and the Ministerial Order of 13 October 1989 (BOE 8) regarding protection of the experimental animals, as well as with the European Council Directive of 24 November 1986 (86/609/EEC).Crystalline NMU  was dissolved in 0.85% NaCl solution and acidified (acetic acid) at 5.0 pH. The concentration was then adjusted to 5 mg/100 g body wt/rat. Heparin-treated blood (0.2 ml) was obtained by cardiac puncture from rats lightly anesthetized with ether.Animals were palpated for detection of mammary tumours initialling after injection of NMU and the animals were examined by palpation for tumour masses thrice weekly post administration of carcinogen. Tumour sizes were measured with a calliper and their growth and histological parameters were tested.The diagnoses of mammary gland cancer were made by histological examination of paraffin sections stained with haematoxylin/eosin in all tumours. Autopsy was performed when the neoplasms were large. A nodule of mammary cancer is designated an active tumour, the number of active tumours is expressed per rat with mammary cancer.The tumour samples were prepared in the following manner: after sacrifice of the animals by decapitation the tumours were removed immediately. All further steps in the preparation were performed at 0–4°C. After being washed in ice-cold PBS buffer (pH 7.4) the tumours were homogenized in this buffer (10 ml/5 g of wet tumour). The homogenate was isolated by centrifugation for 30 min at 9000 × g and the supernatant were stored in aliquots at -80°C until used in each of the corresponding assays.Tumour homogenate and plasma were analyzed for prolactin concentration using a commercially available ELISA kit . The basal level of rat plasma was in accord to that indicated and obtained using standard rat plasma provided by the ELISA kit and indicating that rat stress was avoided, as confirmed by the variability of the plasma blood levels in controls.For TNF α concentration was using a commercially available kit: .An assay for Nitric Oxide concentration was performed according to Navarro-Gonzáles et al .The blood was centrifuged at 1500 g for 15 min; plasma was harvested and stored at -80°C until the hormonal concentration and biomarkers concentration were measured.Total protein concentration was determined by the Lowry assay  for tumour homogenates. In homogenate TNF α, NO concentrations were expressed relative to total protein.Histological analysis was performed on primary NMU tumours on haematoxylin-eosin stained sections. Flow cytometry  was performed of rat NMU induced tumours. Flow cytometry was performed on nuclei prepared from 40 μm thick sections from formalin fixed paraffin tissue by the modified technique of Hedley et al. and McLemore et al ,22.At 55 days of age, the female rats were divided into the following groups:1- Healthy Controls (n = 10) without NMU-induction.2- NMU controls (n = 20) received weekly an injection of NMU [a set of 2 injections during 2 weeks NMU (5 mg/100 g weight/rat)] and was observed after carcinogen administration for tumour development.3- NMU plus metoclopramide (M) (n = 20) in drinking water (0.125 mg/L), "at libitum", was observed weekly for tumour development. The elimination half-life for (M) is 5–6 hours.4- NMU plus goserelin (n = 40) and the metoclopramide administration was suspended in this group when tumour was detected and they were subdivided into 2 groups for (a) acute and (b)chronic study, respectively:a) Received goserelin administration "in bolus"  and killed 2 h after injection.b) Received goserelin (subcutaneous/daily) during 60 days (0.25 mg/ml/day).2 analysis and Yates correction. Inter-group comparisons were performed by Student t test. Values of p < 0.05 were considered to be significantly different.Differences in tumour incidence were demonstrated using xPost administration of NMU in control group, mammary cancer was detected in 9 of 20 (45%) rats on days 90–120. Metoclopramide treatment had effect on NMU-induced tumours, where the incidence was 85%. Latency to the appearance of first palpable tumour tended to be shorter in all rats that received metoclopramide. Indeed the average of tumours was higher than in controls, Table th day, compared with the administered goserelin, where the tumours stop in their development or diminish in size  the size of tumours observed an increase in growth at 400% on 20The histology revealed that all the tumours were adenocarcinoma.Analyses by flow cytometry of nuclear DNA patterns revealed 100% polyploidy in NMU induced tumours. According to this study all tumour samples were polyploidy-aneuploid and none of them was diploid type, percentage of S = 60%, Mitosis (ratio) = 1.99. DNA Index = 1.50 – 2.00, and with a CV (G0/G1) = 9%. Fig plasma the mean value of PRL levels was lower in healthy controls [C]  than in rats with NMU tumours [T] , P < 0.01. During metoclopramide (M) administration the PRL basal mean levels were 125.6 ± 8.5 ng/ml, n = 20 in healthy controls, Fig In The PRL plasma mean levels of NMU tumour rats [T], were higher than in tumour homogenates [TH] (corresponding to the same group of rats and extraction time) 5.8 ± 1.2 ng/ml (n = 20); these results were statistically significant (P < 0.0005).The PRL plasma levels decreased in NMU rats when they were treated with goserelin. During acute administration the time course was, basal: 51.3 ± 2.2 ng/ml (n = 20); 30 min 26.2 ± 6.5 ng/ml (n = 20), P < 0.001; 60 min 17.5 ± 13.5 ng/ml (n = 20), P < 0.0125; and at 90 min: 12.2 ± 4.2 ng/ml (n = 20), P < 0.0005.During chronic administration of goserelin the mean PRL levels were similar to those in healthy control rats: 49.0 ± 23.4 ng/ml (n = 20) P = 0.40, Fig tumour homogenate [TH] there was no statistically significant variations of PRL mean levels during acute administration of goserelin neither post chronically treated: Basal 5.8 ± 1.2 ng/ml (n = 20); 30 min 6.9 ± 0.1 ng/ml (n = 20), P = 0.20; 60 min 5.6 ± 0.3 ng/ml (n = 20); at 90 min 6.8 ± 0.2 ng/ml (n = 20), P = 0.35. At 60 days treatment the mean value was 5.9 ± 0.1 ng/ml (P = 0.35).In plasma. The mean values were: Basal 178.7 ± 11.6 pg/ml (n = 20); at 30 min 150.0 ± 15.0 pg/ml (n = 20), P = 0.10; at 60 min 145.2 ± 2.5 pg/ml (n = 20), P < 0.05; and at 90 min. 135.0 ± 5.0 (n = 20), P < 0.05; Fig TNFα expression during time course of acute administration of goserelin observed inhibition in The inhibition of TNF α expression was observed during post-chronic treatment with goserelin: 132.1 ± 52.2 pg/ml, n = 20 (P < 0.0125).tumour homogenates mean basal values of TNFα were: 255.0 ± 30.0 pg/mg of protein (n = 20), at 30 min 210.0 ± 12.5 pg/mg of protein (n = 18), P = 0.20; at 60 min 200.0 ± 15.0 pg/mg of protein (n = 18), P < 0.05; and at 90 min 210.0 ± 15.0 pg/mg of protein (n = 18), P = 0.10.In At 60 days of goserelin treatment, the TNFα expression observed a statistically significant inhibition when compared with the basal values, 255.0 ± 30.5 vs. 159.0 ± 5.2 pg/mg of protein (n = 18), P < 0.0125, Fig plasma inhibition of NO expression during time course of acute administration of goserelin has been observed but these differences were not statistically significant. Basal mean values were: 59.6 ± 5.5 μg/μl (n = 20), 30 min 56.7 ± 17.8 μg/μl (n = 20), P = 0.10; at 60 min 64.0 ± 17.9 μg/μl (n = 20) P = 0.15 and at 90 min 52.2 ± 7.5 μg/μl (n = 20) P = 0.25. Lack of inhibition of NO expression by goserelin was observed during chronic treatment 46.0 ± 19.9 μg/μl, n = 20 (P = 0.25) when compared with NMU induced basal values, Fig In female Wistar rats without NMU induced tumour, the basal mean NO concentration in plasma was 31.5 ± 9.5 μg/μl, n = 20. The mean NO concentrations were higher in plasma of female Wistar rats with NMU induced tumours than basal healthy controls . In tumour homogenate. The mean basal concentration was: 205.9 ± 42.3 μg/μg of protein (n = 20), at 30 min 134.0 ± 17.9 μg/μg of protein (n = 20) P = 0.05, at 60 min: 118.0 ± 22.9 μg/μg of protein (n = 20), P < 0.05, and at 90 min. 82.3 ± 9.9 μg/μg of protein (n = 20), P < 0.01.The NO concentration was inhibited by goserelin acute treatment in In chronically administered goserelin (at 60 day) the NO values lowered to 72.4 ± 9.9 μg/μg of protein and when compared with basal NO values statistically significant differences was observed between them (P < 0.005), Fig The MNU breast cancer model induced in rats is used for the study of carcinogenesis in mammary cancer, prostate, pancreas, etc. This model is very similar to human neoplastic disease . The devA great variability of various mouse and rat strains for sensitivity on mammary tumours with chemical initiating agents was studied and considered that their results support the need to use more then one rat strain for the initiation of mammary carcinogenesis, due to "very low"-sensitivity for the NMU tumour development in Wistar-Han rats . NeverthOur study demonstrated that metoclopramide (as dopaminergic antagonist) has a very high capacity to induce hyperprolactinemia in rat, indeed the tumour growth accelerated during the observation period and this effect could be influenced or stimulated by PRL release. These results are in agreement with different previous experimental and clinical studies on PRL-induced proliferation. ,27-33. TThe histological study of our tumours revealed adenocarcinoma type, similar to observed in human breast cancer. Flow cytometric S-phase fraction contributes to diagnosis of tumours. At present to our knowledge, only flow cytometric analyses were performed in NMU-induced solid tumours in rat prostate . MonakovInitially an acute administration of LH-RH agonist induces a release of LH and FSH. However, continuous and chronic administration produces an inhibition of the hypophyseal-gonadal-axis though the process of "down regulation" of pituitary receptors for LH-RH. Furthermore the expression on LH-RH receptor gene was established in tumours . In our It is well known that NO is a free radical that mediates a variety of cell functions. The plasmatic NO expression of our study showed a statistically significant increase of NO concentration in rats with NMU-induced tumours when compared to controls, which was observed in basal conditions. These results suggest that NO can stimulate tumour growth. Additionally, TNFα induces iNOS and NO production and could mediate tumour promoter-induced transformation.In this study we have examined PRL, TNF alpha and NO levels in plasma and in tumour homogenates during goserelin administration and, similar behaviour was observed. PRL levels in homogenate presented no statistical significant differences. It could be due to the initial low values in homogenate or because the sensitivity of the assay was insufficient.It is well known that NO levels in cancer cells contribute to tumour angiogenesis and during acute administration of goserelin it did not completely inhibit this increase. At present, the practical importance of these values or relevance with respect to predicting the disease course is not clear.This study demonstrates firstly the role "in vivo" of metoclopramide on tumour growth and secondly for goserelin in NMU-induced mammary tumours in which inhibitory effects were observed on growth as well as with significant tumour regression, besides it also exerted inhibitory effects on plasmatic and tissular PRL, TNFα and ON expression. In summary, these findings could reveal a possible new inhibitory pathway in the tumour development regulation by LH-RH analogues.IV designed and carried out the experiments and wrote the manuscript and REdeS participated in study analysis and drafted the manuscript. All authors read and approved the final manuscript.


L-arginine methyl ester resulted in significant reduction in this apparent 17β-oestradiol-mediated growth promoting effect. Tumours derived from mice receiving 17β-oestradiol-treatment were characterized by a significantly lower fraction of perfused blood vessels and an indication of an increased hypoxic fraction. Consistent with these observations, 17β-oestradiol-treated tumours were less radio-responsive compared to control tumours when treated with a single radiation dose of 15 Gy. Our data suggests that long-term treatment with oestrogen could significantly alter the tumour oxygenation status during breast tumour progression, thus affecting response to radiotherapy.The putative oestrogen receptor negative human breast cancer cell line MDA231, when grown as tumours in mice continually receiving 17β-oestradiol, showed substantially increased growth rate when compared to control animals. Further, we observed that 17β-oestradiol treatment could both increase the growth rate of established MDA231 tumours as well as decreasing the time taken for initiating tumour growth. We have also demonstrated that this increase in growth rate is accompanied by a four-fold increase in nitric oxide synthase activity, which was predominantly the inducible form. Inducible-nitric oxide synthase expression in these tumours was confirmed by immunohistochemical analysis and appeared localized primarily in areas between viable and necrotic regions of the tumour (an area that is presumably hypoxic). Prophylactic treatment with the nitric oxide synthase inhibitor nitro-British Journal of Cancer (2002) 86, 136–142. DOI: 10.1038/sj/bjc/6600032www.bjcancer.comThe Cancer Research Campaign© 2002  L-arginine by a family of isoenzymes called NO synthases (NOS) (in vivo (Nitric oxide (NO) acts as an intercellular secondary messenger in all mammalian organs, participating in a host of functions including vascular homeostasis. It is synthesized from es (NOS) . However enters the target cells and binds to nuclear ER. Following association of the hormone-ER complex with specific DNA sequences (oestrogen response elements), the transcription of a specific set of genes is initiated, leading, among other events, to increased proliferation. This dependence has been well documented with the breast tumour cell line, MCF-7, both in vitro and in vivo  were obtained from Sigma . Tissue culture medium was obtained from GIBCO-BRL  and foetal calf serum was bought from PAA Laboratories . All other reagents were of analytical grade and were purchased from Sigma  or otherwise as indicated in the text.17β-oestradiol pellets  were purchased from Innovative Research of America . NADPH, Dowex-50WX8-400 and NM glutamine and 10% (v/v) foetal calf serum, in an atmosphere of 5% CO2 in humidified air at 37°C.The MDA231 cells were maintained in exponential growth phase in RPMI-1640 medium supplemented with 2 mAll animal procedures were carried out in accordance with the Scientific Procedures Act 1986 and in line with the UKCCCR guidelines 1999, by approved protocols (Home Office Project Licence No. 40-1770).6 MDA231 cells on the back in a volume of 0.1 ml and allowed to grow to about 200 mm3 in volume. Half of animals with size-matched tumours were then treated with E2 pellets  implanted in the scruff of the neck and growth measurements were taken until tumours attained a volume of about 600 mm3. In another set of five animals, E2-treatment was administered 2 days prior to tumour implantation in order to investigate its effect on the onset of tumour growth. Measurements were again taken until tumours attained 600 mm3. All tumours were excised once they attained the required growth size and either fixed or snap frozen in liquid nitrogen for subsequent analysis.Ten female nu/nu mice were injected subcutaneously with 2×10L-NAME was stored frozen at −20°C and added to drinking water and administered ad libitum (1 mg ml−1) to 10 mice. Treatment commenced 2 days prior to tumour implantation and continued while growth was monitored until tumours reached 600 mm3. Half of these animals also received E2 pellets  implanted on the scruff of the neck.M, pH 7.4), sucrose (320 mM), EDTA (100 μM), dithiothreitol (0.05 mM), leupeptin (10 μg ml−1), soybean trypsin inhibitor (10 μg ml−1) and aprotinin (2 μg ml−1). The preparations were then sonicated using an MSE Soniprep 150 for 3×5 s at a nominal frequency of 23 kHz and an oscillation amplitude of between 5 and 10 μm. Samples were placed in ice between each sonication. These suspensions were allowed to stand in ice for a further 10 min, and then centrifuged at 9000 g for 15 min at 4°C. The resultant pellet was discarded and the post-mitochondrial supernatant (cytosol and microsomes) was treated with a strong cation exchange resin (Dowex-50WX8-400) to remove endogenous arginine. The supernatant was incubated with the resin for 5 min and centrifuged at 9000 g for 5 min in order to pellet the resin. This process was repeated twice, after which the cytosol was treated as free of endogenous arginine.Snap frozen tumours were thawed and homogenized (Ultra-Turrax T25 homogenizer) in four volumes of ice-cold buffer containing HEPES  consisted of HEPES buffer , L-valine (50 mM), L-citrulline (100 μM), 10 μML-arginine and 50 μCi ml−1 l-[U-14C]-arginine, tetrahydrobiopterin (10 μM), calmodulin (400 U ml−1), dithiothreitol (2.5 mM), calcium chloride (250 μM), bovine serum albumin (75 mg ml−1) and 1 mM NADPH. The reaction was initiated by the addition of 50 μl of tumour extract (100–300 μg protein) and incubated at 37°C for 10 min. The reaction was terminated by the addition of 5 ml of 50% (v/v) Dowex-50WX8-400 resin in water to bind any remaining arginine. The resin-incubate mix was then left to settle for 20 min before taking an aliquot of the supernatant for analysis by scintillation counting. The enzyme activity associated with iNOS  was measured as a difference in activity carried in the absence and presence of 1 mM ethylene-bis-(oxyethylenenitrilo) tetra-acetic acid (EGTA).Nitric oxide synthase activity was measured by monitoring the conversion of −1 for a cytosol that is 1 mg protein ml−1.ER determination was carried out in tissue cytosol by employing a monoclonal antibody kit . The sensitivity of the system is calculated as the concentration of ER that was distinguishable from the zero standard, i.e., two standard deviations above the zero standard. This was found to correlate to 1.5 fmol ER mg protein−1. After washing off excess primary antibody, this was followed by incubation with a biotinylated bridging antibody and an avidin-biotin complex labelled horseradish peroxidase . Following incubation with 3,3-diaminobenzidine hydrochloride  as a substrate, positive staining for the presence of iNOS was seen as a dark brown end product.Tumours were excised and immediately fixed to provide optimum antigen retrieval and paraffin-embedded for immunohistochemical analysis. Sections were stained with haemotoxylin for cell nuclei. To visualize iNOS localization on sections, an anti-iNOS polyclonal antibody  was employed at a dilution of 5 μg ml3 in volume from control and E2-treated groups were treated with 7-(4′-(2-nitroimidazole-1-yl)-butyl)-theophylline (NITP) 2 h prior to sacrifice (140 mg kg−1 in peanut oil containing 10% DMSO). Hoechst 33342 was next administered 1 min prior to sacrifice . Mice were culled, tumours rapidly excised, snap frozen and stored at −80°C.Tumours that had reached 200 mmImmunohistochemical staining and subsequent analytical procedures were carried out as described elsewhere  was obtained from the log phase of growth. In all treatment groups, regression analysis to calculate r2 values confirmed a linear relationship. To make statistical comparisons between the response of the tumours with or without E2-treatment, the specific growth delay (SGD) was calculated for each tumour using the following equation:Mice were restrained but unanaesthetized during this procedure. Control and Econtrol represents the initial doubling time of the untreated tumours calculated from growth rate attained during exponential growth phase for each tumour type . Tumour doubling times (TD) were calculated from the log phase and linear regression analysis was performed to confirm a linear relationship. Statistical comparisons of differences in mean tumour measurements after treatment between groups were carried out using a two-way analysis of variance (ANOVA) followed by an unpaired Student's 2-treatment on the growth of MDA231 tumours was studied either with treatment commencing prior to tumour implantation . Hence, E2-treatment did not only increase the onset of tumour growth but also increased the growth rate of established tumours. This increase in tumour growth rate following E2-treatment was accompanied by at least a 4–5-fold induction of iNOS activity as determined by the conversion of L-[14C]-arginine to L-[14C]-citrulline .Also represented in ct by E2 . This wa2-treatment groups were below the sensitivity limits of the monoclonal system used which was 1.5 fmol ER mg protein−1  in a region between ‘viable’ tumour cells and true necrotic regions.Confirmation of 3. The results obtained .Multiparameter analysis for tumour hypoxia, vessel density and extent of perfusion were carried out on tumours that had reached 200 mm2-treatment, a single radiation dose of 15 Gy was administered to size-matched tumours at 200 mm3 in size. SGD data was calculated for each treatment group when tumours attained three times treatment size. The data presented . Therefore, the actual NOS activity and consequently NO production within this tumour sub-population, could be much higher than our estimation of NOS activity using whole tumour homogenate. The observed NOS staining in tumour sections was reminiscent of that seen with in situ detection of iNOS mRNA expression in the human colon adenocarcinoma cell line, DLD-1 (2-stimulated increase in iNOS activity may be co-ordinately mediated by the presence of hypoxia. Hypoxia responsive elements (HREs) found within a number of genes involved in energy metabolism and angiogenesis can specifically regulate transcription response to tumour hypoxia . We propose that low and sustained NO production due to E2-treatment in our tumour model provides the angiogenic signal that leads to enhanced tumour cell proliferation. Human tumours in immunodeficient host animals represent a complex model; steroid hormones modulate the residual immune system, influence the endocrine milieu and alter the stroma. E2-treatment could enhance tumour growth by modulating the host to produce other growth factors in vivo (2 on breast cells, may also be produced by stromal cells (2 may cause perturbation of the apoptotic pathway and may be associated with tumourigenesis (Several hypotheses have been put forward to explain the mechanistic basis of E in vivo . It has igenesis .2 growth stimulation, has been the role of NK cell activity. It is widely believed that NK cells have an important role in immune surveillance against tumours (2 (Another area that has received considerable attention in providing a mechanistic basis for Emours (2  and redumours (2 .Initial studies on MDA231 cells ER status led to the understanding that these cells lacked the ability to transcribe the ER gene , now kno2-treated tumours were significantly more radio-resistant when compared to control tumours. In conclusion, the observed alterations in the radiation response due to long term E2-treatment, would suggest that the level of circulating hormone should be taken into consideration in determining the timing and outcome of radiation therapy, particularly to breast cancer patients.The interdependence between NO and hypoxia is believed to play an important role in controlling tumour growth and radiation response. Studies employing NO donor agents have shown that NO sensitizes hypoxic cells to ionizing radiation . It is k


High levels of loss of distal markers on 17p13.3 in breast cancer suggested the presence within the region of at least one tumour-suppressor gene. Here we describe the derivation of two biallelic polymorphisms from the 17p telomeric yeast artificial chromosome (YAC) TYAC98. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and multiplex PCR analysis demonstrated that the high level of allelic imbalance observed in breast tumours represented loss of constitutional heterozygosity (LOH) and that this LOH extended to the telomere. Lung carcinoma (but not Wilms' tumour)-derived DNA again revealed a high level of loss of subtelomeric 17p sequences. Telomeric microsatellite polymorphisms from other chromosome arms did not show such elevated loss in either tumour type. This suggested that the 17p loss observed did not reflect a general telomeric instability and provided further evidence for the presence of a breast cancer tumour-suppressor gene in the distal region of 17p13.3.


This paper deals with the utility of immunoliposomes for the delivery of doxorubicin (DXR) to human ovarian carcinoma cells in vitro and in vivo. We aimed to investigate whether immunoliposome-mediated targeting of DXR to ovarian cancer cells translates in an enhanced anti-tumour effect compared with that of non-targeted DXR liposomes (lacking the specific antibody). Target cell binding and anti-tumour activity of DXR immunoliposomes were studied in vitro and in vivo (xenograft model of ovarian carcinoma). In vitro we observed that target cell binding and cell growth inhibition of DXR immunoliposomes is superior to that of non-targeted DXR-liposomes. However, in vivo, despite the efficient target cell binding and good anti-tumour response of DXR-immunoliposomes, no difference in anti-tumour effect, compared with non-targeted DXR-liposomes, could be determined. The results indicate that premature DXR leakage from immunoliposomes occurring before the actual target cell binding and subsequent DXR association with the tumour cells, explains why no significant differences in anti-tumour activity between DXR-immunoliposomes and non-targeted DXR-liposomes were observed in vivo.


The in vivo antitumour activities of recombinant human interleukin-2 (rHIL-2) and recombinant human hybrid interferon alpha A/D  were tested in relation to adenocarcinoma 755. The tumour growth, following s.c. inoculation of tumour cells, was inhibited to a greater extent in mice treated with the combination of cytokines than in mice treated with either one alone. Pretreatment with these cytokines did not affect the tumour growth. Injection of tumour-bearing mice with a combination of these cytokines resulted in a marked increase in the total number of lymphocytes in the peritoneal cavity. Among them, Lyt-2+/L3T4- and asialo GM1+ cells were markedly enhanced by the combination of cytokines, and the frequencies of these marker cells were closely correlated with the antitumour activity. In tumour-bearing mice, the size of the thymus was decreased while that of the spleen was increased compared to non-tumour-bearing  mice. Treatment with rHIL-2 caused the thymus, spleen and liver to be larger compared to untreated tumour-bearing mice, but when treated with a combination of rHIL-2 and rIFN-alpha A/D these organs were smaller than when rHIL-2 was administered alone. Thymocytes were drastically changed when mice were bearing a tumour or were treated with a cytokine. Especially immature T-cells, Lyt-2+/L3T4+, were drastically decreased in tumour-bearing mice, but were maintained following administration of rHIL-2 or rIFN-alpha A/D. When treated with rHIL-2 plus rIFN-alpha A/D, Lyt-2+/L3T4+ T-cells were decreased while Lyt-2+/L3T4- T-cells were increased. Frequency of immature T-cells, Lyt-2-/L3T4-, was not changed. On the other hand, T-cell subsets of splenocytes were markedly decreased in tumour-bearing mice compared to normal mice, but all the subsets of splenocytes were almost unchanged even when tumour-bearing mice were treated with rHIL-2 plus rIFN-alpha A/D. Thus, injection of rHIL-2 and rIFN-alpha A/D to tumour-bearing mice resulted in induction of Lyt-2+/L3T4- and asialo GM1+ cells in the peritoneal cavity, and the frequencies correlated with the observed antitumour activity in vivo in this murine model. The increase in Lyt-2+/L3T4- T-cells in the peritoneal cavity may be related to changes in the T-cells in thymus.


The monoclonal anti-CEA antibody, A5B7, has previously been administered to patients for radioimmunotherapy (RIT). Long circulation time and the formation of an immune response have limited therapeutic success in the clinic. Antibody fragments can be used to reduce the in vivo circulation time, but the best combination of fragment and radioisotope to use for therapy is far from clear. In this study we have compared the biodistribution of A5B7 IgG and F(ab')2 with chemically cross-linked divalent (DFM) and trivalent (TFM) A5B7 Fab' fragments in nude mice bearing human colorectal tumour xenografts. The cross-linkers were designed to allow site-specific labelling using yttrium 90 (90Y), a high-energy beta-emitter. We have also compared the above antibody forms conjugated to both 131I and 90Y. Both DFM and TFM were fully immunoreactive and remained intact after radiolabelling and incubation in serum at 37 degrees C for 24 h. Biodistribution results showed similar tumour uptake levels and an identical blood clearance pattern for F(ab')2 and DFM with high tumour-blood ratios generated in each case. However, unacceptably high kidney accumulation for both F(ab')2 and DFM and elevated splenic uptake of DFM labelled with 90Y was observed. Kinetic analysis of antigen binding revealed that DFM had the fastest association rate (kass = 1.6 x 10(5) Ms-1) of the antibody forms, perhaps owing to increased flexibility of the cross-linker. This advantage implies that DFM may be more suitable than F(ab')2 radiolabelled with 131I for RIT. TFM cleared from the blood significantly faster than A5B7 IgG when labelled with both 131I and 90Y, producing an improved therapeutic tumour-blood ratio. Kidney accumulation was not observed for [90Y]TFM, but a slightly higher splenic uptake was observed that may indicate reticuloendothelial system (RES) uptake. Overall, tumour uptake was higher for 90Y-labelled antibodies than for 131I-labelled antibodies. Because of the faster clearance, it should be possible to administer a higher total dose of 90Y-labelled TFM than IgG, which is attractive for RIT. Both A5B7 DFM and TFM, therefore, show favourable properties compared with their parent antibody forms.


Anti-tumour responses with CCRG81010, M & B 39565, NSC 353451, 8-carbamoyl-3-(2-chloroethyl)imidazo -1,2,3,5-tetrazin-4(3H)-one (Mitozolomide) in a panel of 4 murine colon tumours of varying growth characteristics and chemosensitivity and a spontaneous murine lymphoma are similar to those seen with standard nitrosoureas. The moderately well differentiated colon adenocarcinoma MAC 16 is nonresponsive to mitozolomide and methylCCNU. Responses in the other 4 lines studied are only achieved near to maximum tolerated dose and at this level there is severe host toxicity. Haemopoietic toxicity is clearly demonstrated by analysis of peripheral blood counts and by CFU-S assays and severe testicular and ovarian toxicity was also seen at dose levels necessary to achieve anti-tumour effects. Using mitozolomide as an example, the study has demonstrated the feasibility of conducting simple but thorough toxicity evaluation for the determination of the therapeutic index. This approach would provide invaluable guidelines for the selection for clinical trial of the most appropriate members of a series of new cytotoxic compounds.


A coupled dynamic behaviour was found early after injection , while a shifted pattern in localisation of hypericin and DiOC18 was seen after 24 h, suggesting that during its migration through the tumour mass, hypericin is released from the lipoprotein complex. In conclusion, we were able to show that the tumour accumulation of hypericin is critically determined by a combination of biological  and physicochemical elements .Hypericin is a potent agent in the photodynamic therapy of cancers. To better understand its tumoritropic behaviour, we evaluated the major determinants of the accumulation and dispersion of hypericin in subcutaneously growing mouse tumours. A rapid exponential decay in tumour accumulation of hypericin as a function of tumour weight was observed for each of the six tumour models investigated, and a similar relationship was found between tumour blood flow and tumour weight. Moreover, there was a close correlation between the higher hypericin uptake in RIF-1 tumours compared to R1 tumours and tumour vessel permeability. To define the role of lipoproteins in the transport of hypericin through the interstitial space, we performed a visual and quantitative analysis of the colocalisation of hypericin and DiOC To confirm these data, a study using C3H mice bearing subcutaneous RIF-1 fibrosarcoma tumours was performed. The tumour drug concentration increased rapidly over the initial hours and peaked (5.5% ID g−1) approximately 6 h after i.v. administration (Photodynamic therapy (PDT) involves the local or systemic administration of a photosensitising drug that, upon light irradiation and in the presence of oxygen, results in tumour destruction . In the A/2 mice . A fast 131I-labelled) and possibly also in magnetic resonance imaging (MRI). In order to better understand the mechanistic background, the present paper addresses basic aspects and principles of the accumulation of hypericin in malignant tissue. Previous results have shown that, compared to normal cells, isolated malignant cells intrinsically do not take up more hypericin labelled derivatives of the compound could be applied in the field of clinical radiodiagnosis, radiotherapy (The following tumour models were used: (a) mouse RIF-1 (radiation-induced fibrosarcoma) cells  subcutaneously (s.c.) grafted in female C3H/Km mice, (b) mouse MH22A hepatoma cells  s.c. grafted in female athymic nude mice, (c) human CaCo-2 colon carcinoma cells (kindly provided by Dr P Augustijns (KU Leuven)) s.c. grafted in female athymic nude mice, (d) human A431 cervix carcinoma cells (obtained from American Type Culture Collection (ATCC)) s.c. grafted in female athymic nude mice, (e) AY27 TCC  rat cells ) s.c. grafted in female athymic nude mice and (f) R1 rhabdomyosarcoma rat cells (kindly provided by Dr W Landuyt (KU Leuven)) s.c. grafted in female athymic nude mice.6) were inoculated s.c. on the depilated lower dorsum of female mice  or B&K Grimston (England)). Tumours were grown to different surface diameters ranging from 2 to 9 mm and to thicknesses ranging between 2 and 5 mm, as measured by a calliper. These dimensions covered tumour weights from ca 5 to 200 mg.Tumour cells . Hypericin (synthesised from emodin anthraquinone according to −1) immediately before injection. Tumour tissues were harvested, weighed and frozen at −20°C until determination of the hypericin content. Similar tissue samples were taken from control mice. Extraction and quantification of tissue hypericin concentrations were performed as previously described , as described  and injected i.p. in tumour-bearing mice (25 mg kg−1). After 48 h, the animals were killed and the dissected and weighed tumour tissues dissolved in 1 ml of tissue solubiliser  at 37°C overnight. The solution was allowed to cool before the addition of 2 ml of ethyl acetate  and 2 ml of 1 N HCl. Absorption of the upper phase was read at 626 nm using a UV/Visible Spectrophotometer . Concentrations were determined from a standard curve of Evans blue dye.The RIF-1 and R1 tumour-bearing animals were used to assess the tumour vessel permeability by a modification of the procedure described by 18  was investigated by i.v. administering hypericin (5 mg kg−1), immediately followed by DiOC18 (5 mg kg−1) to RIF-1 tumour-bearing animals (tumour weight range 150–200 mg) 5 min, 2 h, 6 h or 24 h before being killed. Prior to injection, DiOC18 was suspended (2 mg ml−1) in 40% propylene glycol, 10% ethanol, 4% Tween 80 and water, after which the mixture was sonicated at 60°C for 2 h. In another set of experiments, FITC-dextran  was administered 2 min before killing the RIF-1 tumour-bearing animals that had received hypericin (5 mg kg−1).The intratumoral localisation of hypericin and DiOCμM slices) were taken from each tumour. The first of two serial sections was stained with hematoxylin and eosin (H&E) and the second was examined by fluorescence microscopy ). To specifically visualise hypericin, the Zeiss filter set 14  was used, whereas the distribution of DiOC18 or FITC-dextran was examined with Zeiss filter set 10 .Tumour samples were immediately mounted in medium  and immersed in liquid nitrogen. Different serial cryostat sections (5 μM2 (24 pixels/field) and by measuring the average fluorescence intensity per field for hypericin and DiOC18, respectively. The data were normalised for the maximal fluorescence intensity of each compound, and expressed as percentage fluorescence intensity (% f.i.). From these field-by-field data, scattergrams were constructed with axes representing the % f.i. of each compound. In addition, the absolute field-by-field differences in fluorescence intensity for hypericin and DiOC18 (i.e. Δ=∣(% f.i.hypericin–% f.i.DiOC18)∣) were calculated. The differences were grouped in fractions with increments of 10% and the percentage of fields corresponding to each of the fractions was determined. In total, 15 overlay fluorescence images randomly taken throughout different tumours (n=3) were analysed for each time interval.Overlay fluorescence images were quantitatively analysed using a KS imaging software system  by subdividing the images in 2269 square fields of 38.7 After systemic administration of hypericin, its uptake in tumour tissue was studied as a function of tumour weight in six tumour models. As previous results using the RIF-1 tumour model had demonstrated, a peak concentration of hypericin in tumour tissue between 4 and 8 h after intravenous injection, a 6 h interval between administration and analysis was used in all cases. From To further evaluate the intratumoral and intertumoral differences in hypericin accumulation disclosed in R1 and RIF-1 tumours, we analysed and compared their relative perfusion as well as their vessel permeability as a function of tumour weight. To measure tumour perfusion, FITC-dextran was i.v. injected 2 min before the animal was killed . At this short interval, extravasation of the FITC-dextran complex is negligible . Intratuvs tumour weight, 48 h after administration. Except for small tumours (<20 mg), the relative amount of Evans blue recovered from one tumour type was similar over a large range of tumour weight. However, major intertumoral differences between the permeability of vessels present in RIF-1 and R1 tumours were found.Tumour vessel permeability was measured by i.p. injection of Evans blue dye, a dye with a strong affinity for serum albumin. Extravasation of this dye is based on vessel permeability, which makes it a good marker for permeability measurements  and other lipoproteins  fluorescence are apparent in the vessels and in the perivascular region. Conversely, at 24 h, a more homogeneous distribution is observed for hypericin in contrast to DiOC18-labelled lipoproteins that show an irregular spreading , an identical relationship was found between tumour blood flow and tumour weight. The fact that small tumours are relatively better perfused than larger ones has been documented before .Furthermore, we consistently found that the hypericin uptake in RIF-1 tumours was about twice as high as in R1 tumours. Since this difference cannot be accounted for by a dissimilar tumour perfusion, we investigated the permeability of the tumour vessels involved.6 cells  and high-density lipoproteins (HDL), respectively)  , so thatctively) . Alterna18, a compound that virtually irreversibly associates with lipoproteins. After simultaneous administration of hypericin and DiOC18 to mice bearing RIF-1 tumours, fluomicroscopic analysis of tumour sections revealed that both compounds initially behaved similarly, indicating that hypericin and lipoproteins comigrate. However, later time intervals showed obvious differences in their intratumoral localisation. Thus, while the lipoproteins seem to be limited in their capacity to migrate through the interstitial space, hypericin spreads rather homogeneously over vascularised areas.To investigate which route prevails, lipoproteins were marked with DiOC18, hypericin is an amphiphilic compound that preferentially locates to the polar aprotic zone adjacent to the lipid–water interface (6 was ineffective (These results suggest that during its migration through the tumour mass, hypericin is released from the lipoprotein complex. In support of this notion, it is worth mentioning that unlike the lipophilic DiOCnterface . InteresIn conclusion, the results of the present study indicate that the tumour accumulation of hypericin is critically determined by a combination of tumour-dependent and compound-dependent factors. As far as the tumour tissue is concerned, it is clear that an adequate vascularisation, blood flow and vessel permeability, all contribute to the ability of hypericin to accumulate in the tumour mass. On the other hand, once extravasated and locally delivered by a lipoprotein carrier, the compound itself can dramatically affect the level of its migration throughout the interstitial zone. Our study reveals that hypericin may have an affinity for some constituents typically present in the interstitium, and that there probably is a partitioning of the compound over lipoproteins, structural proteins and lipid bilayers of cancer and immune cells. This partitioning allows hypericin to spread rather homogeneously over the tumour mass.The present study shows that hypericin accumulates less in tumoral sites with a poorly developed or afunctional vascular system, typical of larger tumours. Of importance, since the plasma clearance rate of hypericin is fast, following a two-phase exponential decay with half-lives of only 0.08 and 1.4 h (


The aim of this study was to investigate the effects of application of a methylated cyclic RGD-peptide as an αv-integrin antagonist on angiogenesis, microcirculation, growth and metastasis formation of a solid tumour in vivo. Experiments were performed in the dorsal skinfold preparation of Syrian Golden hamsters bearing the amelanotic hamster melanoma A-Mel-3. Animals were injected intraperitoneally with a methylated cyclic RGD-peptide every 12 h, the control group received an inactive peptide. Microcirculatory parameters of tumour angiogenesis including functional vessel density, red blood cell velocity, vessel diameter and leucocyte–endothelium interaction were analysed using intravital microscopy. In an additional study the effects on growth and metastasis of subcutaneous A-Mel-3 were quantified. Functional vessel density was markedly reduced on day 3 in treated animals compared to controls  and increased subsequently in both groups. Red blood cell velocity at day 3 was below values of controls . No differences were observed in vessel diameters and leucocyte–endothelium interaction was almost absent in both groups. Furthermore, growth and metastasis of subcutaneous tumours after administration of the cyclic RGD-peptide was significantly delayed in comparison to controls (P<0.05). Inhibition of αv-integrins by a cyclic RGD-peptide resulted in significant reduction of functional vessel density, retardation of tumour growth and metastasis in vivo. Taken together, these results implicate RGD-peptides as agents which have anti-tumour and anti-metastatic activity in vivo.Anti-angiogenetic cancer therapy is a potential new form for treatment of solid tumours. The αBritish Journal of Cancer (2002) 86, 788–795. DOI: 10.1038/sj/bjc/6600141www.bjcancer.comCancer Research UK© 2002  Angiogenesis, the growth of new blood vessels from pre-existing vessels, is involved in several physiological and pathological situations like wound healing . For grov-integrins, combined with distinct β subunits, exhibit a high expression in angiogenesis  sequence, including vitronectin, fibronectin, fibrinogen, thrombospondin, proteolysed collagen, von Willebrand factor and osteopontin . The experiments met all the standards required by the UKCCCR guidelines for the welfare of animals in experimental neoplasia ) in accordance with institutional guidelines after approval of the animal committee of the Bavarian state. The animals were housed one per cage and had free access to tap water and standard laboratory food throughout the experiments. Animals were inspected at least two times a day by specialised animal colony staff to assure a normal clinical condition . Particular attention was given to body weight and any signs of discomfort, ulceration or inflammation. All surgical procedures were performed under pentobarbital anaesthesia  and rhodamine 6G-stained leucocytes were visualised using a Leitz N2 filter block . Intravital microscopy was performed 3, 5, 9 and 13 days after implantation of the tumour cells. At least 3–5 sites of interest per animal were randomly selected in centre and periphery of the tumour. Images were acquired by a SIT video camera  and recorded on S-VHS video tape (Sony) for subsequent analysis. Parallel to microvascular observations the area covered by tumour in the chamber preparation was registered on videotape using a Leitz macroscope with video camera  to evaluate tumour growth. The tumour area (mm2) was quantified from videotape by digital image analysis.Quantification of tumour angiogenesis using intravital fluorescence microscopy have been described in detail elsewhere . In brie−1. Red blood cell velocity (vRBC) was quantified with the line shift diagram method according to −1. Rolling leucocytes were defined as population of cells temporarily interacting with the vessel wall and thus having a velocity at least 50% below vRBC in the same vessel. Adherent leucocytes were given as the number of leucocytes remaining stationary for at least 30 s per square millimetre of vessel wall surface . This system described in detail by 6) of the A-Mel-3 were suspended in a 10 μl volume and injected s.c. over the lumbosacral region of the dorsal skin. This is considered to cause the least distress upon the animal because the dorsal skin is extremely expansible. Starting on day 5 after tumour cell implantation, the longer (l) and shorter (w) perpendicular axes and the height (h) of each tumour nodule were measured with callipers every other day. Metastases of the animals were determined by palpation of axillar and inguinal lymph nodes. The day when metastases were first palpable the animal was defined as metastasised. To minimise the pain and distress of the animals no biopsies were taken from the lymph nodes during the observation time. This was due to the known fact that the axillar or inguinal lymph nodes were the most frequent sites of metastasis formation  were anaesthetised with pentobarbital and the dorsal skin was shaven and chemically depilated . Cells (4 to 6×10v antagonist EMD121974 (cyclic Arg-Gly-Asp-D-Phe(N-methyl)Val) . One day after implantation of tumour cells, animals were randomly assigned to two groups (n=6) and RGD-peptide EMD121974 or control-peptide EMD135981 were given i.p. every 12 h (30 mg kg−1 b.w.) until the end of the experiments.The cyclic integrin αU and Kruskal–Wallis test, respectively . Metastasis analysis was performed according to the Kaplan–Meier method . Already on day 5, FVD of the control group reached the maximum value and decreased slightly thereafter until the end of the experiments (day 13). In the treated group the highest value of FVD was observed not before day 9, and stayed nearly at the same level afterwards. On day 13 after tumour implantation, FVD of controls was significantly below values of treated animals .Three days after tumour cell implantation intravital microscopy already revealed an early vascular network in controls  in tumour vessels of treated animals was markedly below values of controls on day 3  and 13  inhibition of angiogenesis at the primary tumour site and thus delayed spreading of tumour cells from the primary tumour and (b) inhibition of angiogenesis in the tumour metastasis, resulting in a delay of growth of tumour metastasis.The growth and metastatic properties of solid tumours are directly influenced by the process of angiogenesis . In earl−1 of the cyclic RGD-peptide twice a day. Plasma half-life time of the cyclic RGD-peptide after intraperitoneal injection was 47 min. However, these data allowed no conclusion on the duration of the peptides' binding to αv-integrins, which may not last the 12 h until next administration. Remarkably, no side effects were observed in treated animals indicating low toxicity of the therapeutic regiment: Body weight increased similar in control and treated animals, and animals showed normal behaviour without signs of any discomfort.Based on previous pharmakokinetic studies, anti-angiogenic therapy was performed by intraperitoneal injections of 30 mg kgWith our second series of experiments we wanted to address the question whether the inhibitory effect on the subcutaneous tumour growth and metastasis may be attributed to changes in tumour angiogenesis and microcirculation. By intravital microscopy, the activity of angiogenesis in tumour tissue can be most accurately quantified by measurement of functional vessel density (FVD), i.e. the total length of perfused microvessels per unit area of observation (v integrin-treatment may be related to continuous delay of tumour growth.Comparable to former observations by v-integrins.Analysis of red blood velocity during tumour angiogenesis revealed differences only at day 3 following tumour implantation. At this time point, control tumours already exhibited a dense vascular network, whereas in tumours following anti-angiogenic treatment few sprouts as signs of early angiogenesis were visible, which contained erythrocytes, but often no movement of red blood cells was seen yet. In contrast to results of studies of the effects of inhibition of VEGF on tumour microvasculature , leucocyv-integrins by a cyclic RGD-peptide resulted in significant reduction of functional vessel density, retardation of tumour growth and metastasis in vivo. The anti-tumor efficacy was associated with remarkable low toxicity of this compound. Administration and dosage of the cyclic RGD-peptide may be further optimised in future studies. Currently, clinical studies are performed to test safety of anti-angiogenic therapy with this cyclic RGD-peptide in humans.Inhibition of α


P<0.0001), while additional cytology on the rinse fluid of the forceps increased the sensitivity to 83.7% (P<0.0001). The addition of both imprint cytology and cytology on the rinse fluid of the forceps increased the diagnostic rate to 86.2% (P<0.0001). Our results indicate that cytological examinations of the imprints of biopsy samples and the rinse fluids of the forceps and the brush improve the efficacy of fibreoptic bronchoscopy in the diagnosis of peripheral lung cancer.To improve the efficacy of fibreoptic bronchoscopy in the diagnosis of peripheral lung cancer, we evaluated the effectiveness of various techniques for obtaining samples for cytological examination. Between January 1984 and December 2000, flexible fibreoptic bronchoscopy under fluoroscopic guidance was performed in 1372 patients with lung cancer having no visible endoscopic findings. Histological examination of specimens obtained by forceps biopsy and cytological examinations on imprints of biopsy specimens, brushing, selective bronchial lavage, curettage, transbronchial needle aspiration, rinse fluids of the forceps, brush, curette, and aspiration needle, and all fluids aspirated during the bronchoscopic examinations were evaluated for diagnostic power. Using these techniques, the overall diagnostic rate with bronchoscopy was 93.4%. The sensitivity of the histological examination was 76.9%; additional imprint cytology increased the sensitivity to 84.8% ( Fibreoptic bronchoscopy is now routinely used for obtaining specimens from which to diagnose various respiratory diseases. Bronchoscopic techniques for the diagnosis of lung cancer consist of histological examination of specimens obtained by transbronchial biopsy and cytological examination procedures such as bronchial washing, brushing, and needle aspiration. Some combinations of these techniques have been reported to increase the diagnostic sensitivity for lung cancer compared with that of transbronchial biopsy alone. Harvesting the cells shed from the tumour during bronchoscopic needle aspiration or brushing, by rinsing the needle or brush, has already been reported to be useful  at the Department of Respiratory Medicine, Okayama Institute of Health and Prevention. Of these patients, 1372 were ultimately given a diagnosis of peripheral lung cancer. The median age were 68 years, ranging from 29 to 90. Altogether, 962 patients were men and 410 women. The histological type of tumour was adenocarcinoma in 812 patients (59.2%), squamous cell carcinoma in 326 (23.8%), small cell carcinoma in 149 (10.9%), large cell carcinoma in 43 (3.1%), other types of carcinoma in 22 (1.6%), and unknown in 20 (1.4%).Among the 1372 patients with peripheral lung cancer, 1212 (88.3%) were given a diagnosis from the results of their first bronchoscopic examination. Furthermore, 64 patients (82.1%) of 78 undergoing the second examinations and all the six patients receiving a third examination had positive results. However, 90 patients could not receive a definite diagnosis by bronchoscopic examination. Accordingly, the overall diagnostic rate by bronchoscopy was 93.4% . Cytological examination of the rinse fluid of the forceps also significantly improved the diagnostic sensitivity, with additional positive results in 94 cases (6.9%) (P<0.0001). In 1332 bronchoscopic examinations that included brushing, cytological diagnosis was given in 764 cases (57.4%). Cytological examination of the rinse fluid of the brush gave additional positive results in 46 cases (3.5%) (P<0.0001).The combination effect of various bronchoscopic techniques is shown in Figure 2P<0.0001), and in 86.2% of cases by biopsy plus imprint cytology plus rinse fluid cytology (P<0.0001).The effectiveness of adding two cytological examinations, on imprints of the biopsy specimens and on the rinse fluid of the forceps, to the histological examination of specimens obtained by forceps biopsy in 1372 bronchoscopic examinations for peripheral lung cancer is shown in Figure 3Among patients who were diagnosed by bronchoscopy, 65 patients with peripheral lung cancer could not be given a diagnosis by the usual bronchoscopic techniques of forceps biopsy and brushing cytology. They were given a diagnosis by cytological examinations of one or more of the following: rinse fluid of the forceps, rinse fluid of the brush, curettage specimens, rinse fluid of the curette, needle aspiration samples, rinse fluid of the needle, all fluid aspirated during bronchoscopy, and selective bronchial lavage samples. Of these 65 patients, 27 received a diagnosis by the following single procedure alone: nine from the rinse fluid of the forceps, nine from curettage specimens, seven from needle aspiration samples, and two from the rinse fluid of the brush.Fibreoptic bronchoscopy has been routinely used for the diagnosis of suspected lung cancer. However, diagnostic results with bronchoscopy for peripheral lung cancer varied markedly among reports: 97.3% by We evaluated the sensitivity of transbronchial biopsy by forceps, imprint cytology of biopsy specimens, brushing cytology, cytology of the rinse fluids of the forceps and brush, and cytology of all fluid aspirated during bronchoscopic examination. In this study, we showed that the combined use of these techniques could increase the diagnostic rate to 93.4%.P=0.009). In the present study, we investigated the effectiveness of imprint cytology instead of the rinse fluid cytology of biopsy specimens, and found that it increased the sensitivity by 7.9 percentage points. Furthermore, we performed cytology examinations on the rinse fluids of the forceps and brush. The effects of such combinations have not been previously reported. The rinse-fluid cytology of the forceps increased the sensitivity from 76.9 to 83.8% (P<0.0001) and the rinse fluid cytology of the brush increased the sensitivity from 57.4 to 60.9% (P<0.0001). Therefore, we considered that the various cytological procedures that we used during bronchoscopy were highly useful for improving its effectiveness in the diagnosis of peripheral lung cancer.Cytological examination of the rinse fluids of bronchial biopsy specimens was first mentioned by Regarding the histological type of lung cancer, Similarly, no difference in diagnostic rate by tumour size was found in this study. The diagnostic rate of each technique was less than 80%; however, it was increased to 93.4% by combining all the techniques. Furthermore, 27 of the 1372 patients (2%) were given a diagnosis by only one technique, such as cytological examination of the rinse fluid of the forceps or brush. These results suggest that using a variety of techniques has a valuable complementary role in the diagnosis of lung cancer.When the diagnosis for peripheral lung tumour was not obtained by the first bronchoscopic examination, the diagnostic rate was raised by repeating the examination . HoweverIn conclusion, we recommend that the inexpensive and efficient bronchoscopic techniques of imprint cytology of biopsy specimens and cytology of the rinse fluids of the forceps and brush be routinely performed in the diagnosis of peripheral lung cancer.


All three dye preparations induced complete EMT-6 tumour regression in 75–100% of animals at a low drug dose (0.25 μmol kg−1) following PDT  at 24 h pi. Complete tumour regression in the Colo-26 tumour model was obtained in 30% of mice at a dose of 2 μmol kg−1. In the non-cured animals, AlPc–PVA induced the most significant tumour growth delay. This dye showed a prolonged plasma half-life (6.8 h) as compared to AlClPc (2.6 h) and AlPc–PEG (23 min), lower retention by liver and spleen and higher tumour-to-skin and tumour-to-muscle ratios. Our data demonstrate that addition of hydrophilic axial ligands to AlPc, while modifying in vitro and in vivo kinetics, does not reduce the PDT efficiency of the parent molecule. Moreover, in the case of the polyvinylalcohol derivative, axial coordination confers advantageous pharmacokinetics to AlPc, which makes this photosensitizer a valuable, water soluble candidate drug for clinical PDT of cancer. © 1999 Cancer Research CampaignThe potential use of unsubstituted aluminium phthalocyanine (AlClPc) as a sensitizer for photodynamic therapy (PDT) of cancer has not been fully exploited in spite of its higher efficiency as compared to the sulphonated derivatives. This is largely due to the strong hydrophobic character of AlClPc which renders the material difficult to formulate for in vivo administration. We prepared two water-soluble derivatives of AlClPc by axial coordination of polyethyleneglycol  or polyvinylalcohol  to the central aluminium ion. Their photodynamic activities were evaluated in vitro against the EMT-6 mouse mammary tumour cells and in vivo against the EMT-6 and the colon carcinoma Colo-26 tumours implanted intradermally in Balb/c mice. Pharmacokinetics were studied in the EMT-6 tumour-bearing mice. After 1 h incubation, the light dose required to kill 90% of cells (LD


Patients with small-cell lung cancer who survive more than 2 years have a significantly increased risk (relative risk of 3.6) of developing a second primary tumour. The cessation of cigarette smoking after successful therapy is associated with a significantly decreased risk of a second primary tumour.


However, the maximum tolerated dose of 5,6-dimethylxanthenone-4-acetic acid was considerably higher in tumour necrosis factor receptor-1−/− mice (>100 mg kg−1) than in wild-type mice (27.5 mg kg−1). The antitumour activity of 5,6-dimethylxanthenone-4-acetic acid (25 mg kg−1) was strongly attenuated in tumour necrosis factor receptor-1−/− mice. However, the reduced toxicity in tumour necrosis factor receptor-1−/− mice allowed the demonstration that at a higher dose (50 mg kg−1), 5,6-dimethylxanthenone-4-acetic acid was curative and comparable in effect to that of a lower dose (25 mg kg−1) in wild-type mice. The 5,6-dimethylxanthenone-4-acetic acid -induced rise in plasma 5-hydroxyindoleacetic acid, used to reflect serotonin production in a vascular response, was larger in colon 38 tumour bearing than in non-tumour bearing tumour necrosis factor receptor-1−/− mice, but in each case the response was smaller than the corresponding response in wild-type mice. The results suggest an important role for tumour necrosis factor in mediating both the host toxicity and antitumour activity of 5,6-dimethylxanthenone-4-acetic acid, but also suggest that tumour necrosis factor can be replaced by other vasoactive factors in its antitumour action, an observation of relevance to current clinical studies.5,6-dimethylxanthenone-4-acetic acid, a novel antivascular anticancer drug, has completed Phase I clinical trial. Its actions in mice include tumour necrosis factor induction, serotonin release, tumour blood flow inhibition, and the induction of tumour haemorrhagic necrosis and regression. We have used mice with a targeted disruption of the tumour necrosis factor receptor-1 gene as recipients for the colon 38 carcinoma to determine the role of tumour necrosis factor signalling in the action of 5,6-dimethylxanthenone-4-acetic acid. The pharmacokinetics of 5,6-dimethylxanthenone-4-acetic acid, as well as the degree of induced plasma and tissue tumour necrosis factor, were similar in tumour necrosis factor receptor-1British Journal of Cancer (2002) 87, 465–470. doi:10.1038/sj.bjc.6600479www.bjcancer.comCancer Research UK© 2002  The tumour vasculature is a promising new target in cancer therapy, and a number of antitumour agents have been identified that either inhibit tumour angiogenesis . Tumour In situ hybridisation studies indicate that both host and tumour cells within murine colon 38 tumours express TNF mRNA after DMXAA treatment  as recipients for the (TNF positive) colon 38 carcinoma. We have examined four end-points, toxicity, tumour growth delay, induction of tumour haemorrhagic necrosis, and serotonin production. Because DMXAA pharmacokinetics are of major importance in the interpretation of experiments combining DMXAA with drugs such as thalidomide (−/− mice.The activities of TNF are mediated by binding to two receptors: TNFR1 (p55), and TNFR2 (p75) . TNFR1 i−/− mice on a C57Bl background were obtained from Jackson Laboratories, Bar Harbor, Maine, USA. WT and TNFR1−/− mice were bred in the Animal Resources Unit, University of Auckland (TNFR1−/− mice under specific pathogen-free conditions). All experiments were approved by the University of Auckland Animal Ethics Committee, and conformed to the Guidelines for the Welfare of Animals in Experimental Neoplasia, as set out by the United Kingdom Co-ordinating Committee on Cancer Research. Mice were used between 6 and 12 weeks of age and the colon 38 tumour was implanted subcutaneously in mice that had been anaesthetised by intraperitoneal (i.p.) administration of pentobarbitone (87 mg kg−1). DMXAA was synthesised in this laboratory (−1 body weight).Breeding stocks of C57Bl/6 and TNFR1a2×b, where a and b were the minor and major tumour axes. The arithmetic means and standard error of the means were calculated for each time point, counting cured animals as having zero tumour volume, and expressed as fractions of the pre-treatment tumour volume. Growth delay was determined as the difference in the number of days required for the untreated and treated tumours to reach four times the pre-treatment volume. Tumour necrosis was measured in WT and TNFR1−/− mice in which colon 38 tumours had been implanted and allowed to grow to a diameter of approximately 6 mm. Tumours were removed 24 h after drug treatment, fixed in 10% formalin, sectioned and stained with haematoxylin/eosin. Tumour necrosis was quantified microscopically using a grid system  the serum was then removed and stored at −70°C. Tumour, spleen and liver tissues were homogenised in 1.5 ml of α-MEM medium using a tissue homogeniser. The homogenates were centrifuged  and the supernatant was removed and re-centrifuged . Serum and supernatants from tissue homogenates were kept at −70°C until use. TNF was assayed using a commercially available ELISA kit  according to the manufacturer's directions.Mice were anaesthetised with halothane and bled from the ocular sinus. The blood was allowed to coagulate overnight on ice. After centrifugation of samples (2000 M HCl containing 0.01% ascorbic acid. 5HIAA concentrations were measured using automated solid phase extraction and high performance liquid chromatography (HPLC) as previously described (Blood samples (700–800 μl) from DMXAA-treated and untreated mice were collected from the ocular sinus into heparinised tubes during halothane anaesthesia, centrifuged, and the plasma was removed and mixed with 0.1 −1 i.p.). After 0.25, 1, 2, 3, 8 and 24 h, blood samples (700–800 μl) were collected from the ocular sinus into heparinised tubes during halothane anaesthesia, centrifuged, and the plasma was removed and stored at −20°C until analysis. DMXAA plasma concentrations were measured using automated solid phase extraction and high performance liquid chromatography (HPLC) as previously described (Tumour-bearing mice (three per group) were treated with DMXAA  of DMXAA in TNFR1−/− mice were compared. In WT mice, DMXAA (25 and 27.5 mg kg−1) produced growth delays of 13 and 19 days, respectively, and cure rates of 40%  induced only a short tumour growth delay (4 days) with no cures. However, a higher dose (50 mg kg−1) induced a tumour growth delay of 13 days and a cure rate of 60% . Tumours from untreated WT and TNFR1−/− mice showed either little necrosis or central necrosis occupying up to 30% of the section area. Tumours from DMXAA-treated WT and TNFR1−/− mice showed extensive areas of haemorrhagic necrosis that were identical in histological appearance. The proportion of tumour haemorrhagic necrosis in mice treated with DMXAA (25 mg kg−1) was 99% in WT and 76% in TNFR1−/− mice, but at higher doses, tumours in TNFR1−/− mice exhibited a similar degree of necrosis (Colon 38 tumours growing in WT and TNFR1necrosis .−1) to non-tumour bearing WT mice produced a significant (P<0.001) increase in 5HIAA after 30 min, followed by a further increase to a maximum after 4 h and a decrease thereafter. Administration to non-tumour TNFR1−/− mice also produced a significant increase in 5HIAA after 15 min (P=0.001) and 60 min (P<0.001) but no further increase thereafter   were 1623 and 1609 μmol.h l−1, respectively.To determine whether the difference in antitumour activity of DMXAA in WT and TNFR1−/− mice , indicating a TNF-independent vascular effect and consistent with the hypothesis that DMXAA can cause direct damage to the vascular endothelium  is sustained for at least 24 h, consistent with the presence of an extended, tumour-specific vascular response. This sustained response is lacking in TNFR1−/− mice treated at the same dose, in agreement with the reduced induction of tumour haemorrhagic necrosis and smaller induced tumour growth delay (−/− mice (−/− mice at high dose.The 5HIAA responses of tumour-bearing TNFR1th delay ; Table 2−/− mice , consist−1, DMXAA demonstrates excellent antitumour activity in TNFR1−/− mice without host toxicity (In conclusion, the demonstration that at a dose of 50 mg kgtoxicity ; Table 1toxicity , interletoxicity  and nitr


A method for determining the tumour cell content of breast neoplasms by morphometry is described.3, or 21·5% of the entire neoplasm. Two medullary lesions gave a mean tumour cell volume of 417 mm3, 64·5% of the total volume of the neoplasm.The mean total tumour cell volume of 10 scirrhous carcinomata was 287·6 mmA possible extension of this technique to monitor changes in the character of malignant tumours is discussed.


The prognostic significance of tumour cell proliferation was investigated in a series of 418 gastric carcinomas using the monoclonal antibody MIB-1. Owing to strong intratumoural heterogeneity of MIB-1 expression three different proliferation indices (PIs) were determined in all carcinomas: (1) PImax in areas of maximal tumour cell proliferation, (2) PIrand in areas randomly distributed over the whole tumour. (3) PIfront in areas exclusively located at the tumour invasion front. There was a strong intertumoral heterogeneity with PImax ranging from 4.9% to 92.2%, PIrand ranging from 3.4% to 81.4% and PIfront ranging from 4.2% to 87.1%. The mean values were 51.3% +/- 19.7 for PImax, 34.2% +/- 18.3 for PIrand and 37.2% +/- 19.5 for PIfront. Whereas no statistically significant correlation could be found between proliferative activity and the clinicopathological parameters depth of invasion, lymph node involvement or grade of tumour differentiation, there was a positive correlation between a high proliferation index at the tumour invasion front (PIfront) and the presence of blood or lymphatic vessel invasion. No significant correlation could be demonstrated between the different proliferation indices and survival, even when different subgroups of patients were analysed separately. The present results suggest that the immunohistochemical evaluation of the proliferation activity has no predictive value for the prognosis of gastric cancer patients or the identification of subgroups of patients who may be at higher risk.


At 1, 2 or 4 weeks after tumour transplantation, the mice were bled to obtain plasma and then challenged with 25 micron E. coli lipopolysaccharide (LPS) endotoxin i.v. The mice were killed 24 hr later, further plasma was obtained and their liver ratios and spleen ratios were determined. A similar procedure was carried out on non-tumour-bearing mice. Progressive tumour growth was associated with an increase in the liver ratio. In parallel, mice with 4-week tumour transplant showed increased uptake of colloidal carbon particles and 51Cr-labelled sheep red blood cells in the liver. The plasma amino aspartate transaminase (AST) and the ornithine carbamoyl transferase (OCT) showed a constant rise in all groups of mice after LPS injection. However, at 24 hr after LPS injection, the AST level showed the greatest rise in mice with 4-week tumour transplants. By contrast, OCT, which is liberated only from hepatocytes, showed the greatest rise in non-tumour-bearing mice.


Iodised oil (lipiodol) administered via the hepatic artery localises selectively in primary liver cell cancers (hepatocellular carcinomas or HCCs) for prolonged periods and has been used as a vehicle for cytotoxic agents. Despite clinical use, the mechanism of lipiodol retention by tumours has remained unclear, embolisation of oil droplets in the tumour vasculature being the prevailing hypothesis. We have investigated the role of tumour and endothelial cells in lipiodol retention. Human liver tumour (Hep G2) cells and human umbilical vein endothelial cells in culture were exposed to lipiodol. Light microscopy using selective silver impregnation stains and transmission electron microscopy revealed lipiodol incorporation by both cell types, probably by pinocytosis. This was not associated with cellular injury in terms of cell lysis, cell replication or radio-labelled leucine uptake. Histological analysis of 24 HCCs either surgically resected or discovered incidentally at liver transplantation  revealed vesicles of lipiodol in the cytoplasm of tumour cells and endothelial cells lining tumour vessels. Thus, lipiodol is likely to deliver cytotoxic agents directly into tumour cells and endothelial cells, both in vitro and in vivo. This may also apply to other lipids and to other human tumours. These findings have significant therapeutic implications.


Establishing clinically relevant animal models of glioblastoma multiforme (GBM) remains a challenge, and many commonly used cell line-based models do not recapitulate the invasive growth patterns of patient GBMs. Previously, we have reported the formation of highly invasive tumour xenografts in nude rats from human GBMs. However, implementing tumour models based on primary tissue requires that these models can be sufficiently standardised with consistently high take rates.In this work, we collected data on growth kinetics from a material of 29 biopsies xenografted in nude rats, and characterised this model with an emphasis on neuropathological and radiological features.in vivo, whereas only one of four lower grade tumours engrafted. Average time from transplantation to the onset of symptoms was 125 days ± 11.5 SEM. Histologically, the primary xenografts recapitulated the invasive features of the parent tumours while endothelial cell proliferations and necrosis were mostly absent. After 4-5 in vivo passages, the tumours became more vascular with necrotic areas, but also appeared more circumscribed. MRI typically revealed changes related to tumour growth, several months prior to the onset of symptoms.The tumour take rate for xenografted GBM biopsies were 96% and remained close to 100% at subsequent passages In vivo passaging of patient GBM biopsies produced tumours representative of the patient tumours, with high take rates and a reproducible disease course. The model provides combinations of angiogenic and invasive phenotypes and represents a good alternative to in vitro propagated cell lines for dissecting mechanisms of brain tumour progression. In tumour biology, model systems typically involve cancer cells that have been passaged numerous times ll lines . In animll lines  to produll lines ,4. Notabll lines -8. Thus,in vitro [Orthotopic models are characterised by tumour growth in the animal, in a site corresponding to the human organ in which the tumour arises -11. Therin vitro . Indeed,in vitro -18, and in vitro .Previously, we established an orthotopic biopsy-based model to study angiogenesis and malignant progression . These eHowever, experimental tumour models must be reproducible as well as representative. Since orthotopic models are technically challenging, and since primary tumour tissue display more cellular heterogeneity than cell lines, their use has been limited. Therefore, data demonstrating sufficiently high take rates and a reproducible disease course is needed before we can implement these models to their full potential. In order to address these issues, we studied tumour growth in a consecutive series of 29 glioma biopsies used in xenograft implantations. The animals were scanned using MRI, survival was registered, and brains were examined. This series also included the xenograft tumours we previously used to investigate the invasive and vascular phenotypes in brain tumours . HoweverTumour biopsy tissue was obtained from patients at the neurosurgical department at Haukeland University Hospital, Bergen. The study was approved by the regional Ethical Board at Haukeland University Hospital. Spheroids were prepared as described previously .ad libitum. The athymic nude rat is T-cell deficient, but has normal complement and B-cell function [in vitro. Animals were sacrificed at the onset of symptoms using CO2 and the brains were removed. All procedures and experiments involving animals in this study were approved by The National Animal Research Authority and conducted according to the European Convention for the Protection of Vertebrates Used for Scientific purposes.Studies were conducted with 197 male and female homozygous nude rats (Han:rnu/rnu Rowett) bred and maintained in an isolation facility in a pathogen free environment on a standard 12/12 h day and night cycle. Animals were fed a standard sterilised pellet diet and provided sterile tap water function . 10 tumoPatient MRI images were obtained by using a MRI Magnetom Vision Plus 1.5 T scanner . Animals engrafted with tissue from the initial 9 biopsies were imaged, using the same MRI combined with a small circular finger coil . Subsequ2O2 . The fraction of labelled tumour cells, defined as the Ki67 labelling index (Ki67 LI), was assessed in 5 microscopic high power fields, appearing as "proliferative hot-spots", which contained a high fraction of labelled cells.Brains were immersion fixed in 4% formaldehyde in Dulbecco's Phosphate-Buffered Saline for 24 h. The brains were then embedded in paraffin, and 5 μm sections were prepared. Every 20th section was collected for further histological analysis. These sections were stained with haematoxylin and eosin, and examined by a neuropathologist under a light microscope. Representative formalin-fixed, paraffin-embedded sections from each specimen were immunohistochemically stained with Ki67 ) or human specific nestin (1:500) antibodies  by use of the ABC method and the DCS Detection-Kit with 3, 3"-Diaminobenzidine (DAB) and HThe cell cycle distribution of the tumours was determined by flow cytometric DNA analysis. The tumour tissue was minced, fixed in ice-cold 70% ethanol and washed in 0.9% NaCL and then incubated at 37°C for 15 min with 0.5% pepsin  in 0.9% NaCl solution (pH1.5). The nuclear suspension was exposed to 100 μl of 1 mg/ml RNAse for 1 min, and then stained with propidium iodide . All samples were filtered through a 60 mm nylon filter before analysis with a FACSort flow cytometer . Gating was performed from a two-parameter intensity diagram (FL2 width/FL2 area) in order to exclude doublets and triplets within the fluid flow. Determination of the relative proportions of the cell cycle distribution was obtained using the FACS software platform.Tissue sections were de-paraffinised and the epitopes were unmasked with protease K  for 10 min at room temperature. Detection of apoptotic cells was performed with the terminal deoxynucleotidyltransferase mediated nick end labelling (TUNNEL) assay according to the manufacturer's instructions . TUNNEL -positive cells were visualised with DAB as chromogen, and the fraction of Tunnel-positive cells was assessed from 5 high power fields.A p-level of ≤ 0.05 was considered significant. We used the two-tailed Student's t-test for comparison and Spearmans' test to correlate culture time and patient age with survival time and take rates. We used log-rank test to analyse survival data. A p-level of ≤ 0.05 was considered significant.in vitro in agar-overlay cultures as previously described [We xenografted biopsies from 29 glioma patients, of which 25 were diagnosed with GBM, 3 patients had an astrocytoma grade II, and one had an oligoastrocytoma grade III Table . None ofescribed  and implin vitro similar to the human biopsies. The resulting tumour spheroids were re-implanted into a new generation of nude rats and serially passaged for up to 19 generations  non-angiogenic and invasive tumours, 2) angiogenic and invasive tumours and 3) angiogenic and poorly invasive tumours  was 9.3 days, which compares well to other cell line based studies -33. Morein vivo.Furthermore, MRI provided information about essential aspects of tumour growth such as tumour volume, contrast enhancement, invasiveness, shift of midline structures, surrounding oedema as well as hydrocephalus. Based on these features, MRI reliably distinguished the different tumour phenotypes identified by histological examination. Thus, MRI with clinical sequences can be used in used in conjunction with this model to distinguish the different tumour phenotypes by and monitor growth by imaging Histological analysis at day 3 after implantation revealed that the implantation site contained few cells in the centre, but with cellular infiltration and microhemorrhages at the periphery. This picture may reflect initial reactive changes following implantation, where most tumour cells fail to adapt to the new environment and die. Accordingly, the small tumour visible in the implantation site after 7 days reflects a regrowth of the tumour cells that survive the initial phase. The extensive spread of tumour cells after 30 days suggest that activation of pro-invasive programs is an integral part of the adaptive behaviour needed to sustain growth during the early phases.In the late stages, at the onset of symptoms, the first generation tumours recapitulated the invasive features of the parent tumours like previously reported, but were largely devoid of angiogenesis and necrotic areas. Moreover, tumour cells were found in more than two cerebral lobes and in the contralateral hemisphere. The morphology of tumour cells at high magnification was similar to that observed in patient biopsies.in vivo, pointing to an additional mechanism for the increased overall growth rates that we observed in higher generation tumours. Thus, the faster growing high generation tumours result both from increased gain due to higher cell proliferation, as well as reduced cell loss due to less apoptosis.In contrast, high generation tumours fulfilled all diagnostic criteria of human GBMs, but grew less invasive. The tumours had a higher cell density and the tumour cells displayed a more malignant phenotype compared to low generation tumours and patient biopsies with large, polymorphic nuclei and a less fibrillary, but more circumscribed cytoplasm. Immunohistochemistry also showed that the Ki67 labelling was higher in the tumour xenografts than in the patient biopsies, and increased with passaging. Moreover, tunnel analyses shows that cell death rates declined with propagation in vivo passaging provides a model for modulating essential brain tumour phenotypes. Since these different phenotypes can be derived from the same patient, variation in genetic background due to inter-individual differences is eliminated. Further, systematic comparisons of angiogenic and non-angiogenic tumours that are both invasive and otherwise similar may help identify genes and proteins critical to the onset and maintenance of brain tumour angiogenesis. Using the same strategy on invasive and non-invasive tumours can help to identify invasion-related genes and proteins [It is an important question whether this progression seen in the rat is also the course of primary GBMs in the patients. It is conceivable that the initial GBM in the patient is growing as an invasive lesion without angiogenesis. GBMs are mostly diagnosed at late stages, when the tumours have reached a certain size causing symptoms and changes in MRI due to mass effects, angiogenesis with breakdown of the blood-brain-barrier and necrosis. Thus, early stages are not easily detectable on contrast enhanced sequences, due to lack of these features. While we previously demonstrated that passaging the tumours for 4-5 generations was accompanied by an onset of angiogenesis , this stproteins . We therIn conclusion, our data demonstrate that xenograft tumours representative of the parent tumour can be established in nude rats with a high take rate and a reproducible disease course. Furthermore, MRI using standard clinical sequences provides useful information regarding critical GBM phenotypes involved in tumour progression. Studies at various time points as well as at different stages of passaging suggest that the adaptive behaviour of these tumours display to main phases; one initial phase relying on pro-invasive programmes, and one later stage characterised by angiogenesis, increased cell proliferation and reduced apoptosis, leading to increased overall growth rates. In humans, this model may have a special relevance to early stages of tumour growth, prior to onset of symptoms and diagnosis.The authors declare that they have no competing interests.JW carried out tissue culture, animal experiments and MRI scanning, participated in study design and manuscript preparation. HM, PCH, NB, HG and SW carried out histological examination and immunohistochemistry staining. PS helped manuscript editing. XL and PZ helped animal experiments. MC performed tunnel staining. RB helped in flow cytometry, participated in study design. PE helped statistical analysis, participated in study design and manuscript preparation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/465/prepub


Reactivation of the varicella zoster virus (VZV) is more common in patients with malignancies; however, the molecular and cellular mechanisms underlying this susceptibility are unclear.ex vivo interferon-γ ELISpot assays, we set out to analyse VZV-specific immune responses in a large cohort of patients with malignancies.Using We observed that patients with malignancies had impaired VZV-specific T-cell responses, particularly in those with haematological malignancies and breast carcinoma. Immediate-early protein 63 (IE63)-specific T-cell responses were significantly impaired in those with subclinical VZV re-activation.Our results suggest that T-cell responses to IE63 are important in controlling VZV replication. Malignancy is associated with reactivation of chronic persistent viruses such as varicella zoster virus (VZV) , causingVZV glycoproteins I and E, immediate-early protein 63 (IE63) and four specific CD4+ T cells have been shown to circulate at persistently high frequencies in the peripheral blood of healthy seropositive donors without a history of reactivation . HoweverFresh heparinised venous blood samples were obtained from 106 adult individuals with malignancies (before receiving chemotherapy) who were admitted to the Cancer Institute in Sri Lanka with a past history of primary VZV infection. Samples were also obtained from 26 patients after chemotherapy. Ethical approval for the study was obtained from the local ethics committee.The mean age of the donors was 49.81 years (s.d.±17.5) and 55 (51.88%) were males. The mean age at which the patients had contracted primary VZV was 18 years (s.d.±12.67). In all, 88 patients (83.01%) had a solid malignancy (SM) and 18 (16.9%) had a haematological malignancy (HM) . The heaEx vivo ELISpot assays were performed as previously described , Flu and CMV viruses and have been used for quality control in ELISpot assays  . Data were analysed using nonparametric tests, as frequency distribution curves showed that the data were not normally distributed. Wilcoxon's matched-pairs test was used to determine the P<0.0001) in patients with malignancies  when compared with healthy VZV seropositive individuals . However, there was no difference between the two groups for gE or IE63 overlapping 20-mer peptides (P<0.0001). The overall VZV-specific T-cell responses in individuals with different malignancies are shown in P<0.05) total white cell count  and significantly higher (P<0.005) lymphocyte count  than patients with solid malignancies. However, we found no correlation with the T-cell response with either total white cell or lymphocyte counts. We also observed no correlation between VZV-specific T-cell responses and age of the individual or time since occurrence of primary infection.We observed that overall VZV-specific T-cell responses were significantly diminished . Furthermore, we found that there were no significant differences in the overall VZV-specific functional T-cell responses or for responses to gE or IE63 overlapping peptide pools in this group of patients. VZV-specific T-cell responses also did not significantly differ between patients on different chemotherapy regimes.P<0.01) overall VZV-specific T-cell responses  than those with no reactivation . Furthermore, responses to VZV IE63 overlapping 20-mer peptides were significantly lower (P<0.005) in those with subclinical reactivation  than in those with no reactivation . There was no difference in T-cell responses in these two groups for VZV gE overlapping 20-mer peptides  patients. The viral loads in these patients varied from 30.93 to 198 viral copies per ml of blood . We observed no viral reactivation in the healthy controls. Overall, those who had subclinical VZV reactivation had significantly lower  of the patients. Interestingly, overall mean VZV vaccine-specific T-cell responses, and responses to IE63 overlapping 20-mer peptides were significantly lower in those with VZV reactivation when compared with other patients, suggesting that IE63-specific T-cell responses could be important in the control of VZV replication. However, it will clearly be important to extend these findings to other viral proteins.We failed to see any significant differences between VZV-specific T-cell responses in our patient cohort in both before and after chemotherapy. However, as post-chemotherapy samples were obtained 1–2 weeks after the initiation of chemotherapy, this might not suffice for the occurrence of immunosuppressive effects of the chemotherapy. Therefore, it is important to expand this study in a larger population of patients who received chemotherapy and who were followed up for longer periods.In summary, we have shown that a large cohort of patients with malignancies had impaired T-cell responses to VZV and that virus-specific T-cell responses were lowest in those with subclinical virus reactivation. Interestingly, IE63-specific T-cell responses were significantly impaired in those with subclinical VZV reactivation, suggesting that T-cell responses to IE63 could be important in controlling VZV reactivation. Furthermore, the data suggest that enhancement of IE63-specific T-cell responses may prove beneficial in future vaccination strategy development.