There are no known molecular biomarkers of local recurrence. We searched for β-catenin gene was performed for 155 frozen fibromatosis tissues from all topographies. Correlation of outcome with mutation rate and type was performed on the extra-abdominal fibromatosis group (101 patients).Direct sequencing of exon 3 β-catenin were detected in 83% of all cases. Among 101 extra-abdominal fibromatosis, similar mutation rates (87%) were observed, namely T41A (39.5%), S45P (9%), S45F (36.5%), and deletion (2%). None of the clinico-pathological parameters were found to be significantly associated with β-catenin mutational status. With a median follow-up of 62 months, 51 patients relapsed. Five-year recurrence-free survival was significantly worse in β-catenin-mutated tumours regardless of a specific genotype, compared with wild-type tumours .Mutations of β-catenin mutation hallmarks extra-abdominal fibromatosis from a large multicentric retrospective study. Moreover, wild-type β-catenin seems to be an interesting prognostic marker that might be useful in the therapeutic management of extra-abdominal fibromatosis.A high frequency (87%) of  Fibromatoses are rare, locally invasive soft-tissue tumours composed of spindle cells that display a wide spectrum of aggressiveness. These star-shaped soft tissues, devoid of a capsule, invade surrounding structures and have a high propensity for local recurrence .Clinical forms of the disease depend on the tumour site: abdominal fibromatosis (deep or desmoid), which occurs on the abdominal wall of women in association with hormonal factors; intra-abdominal fibromatosis, some forms of which are associated with germ-like mutations of the adenomatous polyposis coli (APC) gene-associated Gardner syndrome; and extra-abdominal fibromatosis, which occurs in the head and neck, trunk, and limbs. Sporadic extra-abdominal fibromatosis has a high tendency for local recurrence, even after apparently adequate resection. In institutional retrospective studies, local failure rates at 5 years have ranged from 25 to 60%  signalling pathway. The phosphorylation of β-catenin on threonine 41 and serine 33, 37, and 45, encoded by exon 3 of the β-catenin gene, occurs when β-catenin binds to APC and leads to protein degradation through the ubiquitin–proteasome pathway  that might help oncologists in the clinical management of patients with extra-abdominal fibromatosis have been identified. From a biological point of view, desmoids and extra-abdominal fibromatosis tumours typically harbour mutations of APC and/or Conticanet in France, Belgium, and Switzerland to evaluate whether β-catenin mutational status might constitute a valuable biomarker for recurrence that could improve the therapeutic management of such disabling tumours.In this study, we analysed a large series of extra-abdominal fibromatoses (with follow-up data spanning over 5 years) collected through the connective tissue cancer network μm-thick tumour slices flanked by 4-μm sections from either frozen or FFPE fibromatosis tumours for use as histological controls (haematoxylin–eosin–saffron staining). The histological controls were analysed by pathologists specialised in soft-tissue tumours (Ph T and JMC). Over 90% of the histological features identified were typical of fibromatosis in all tumours analysed.All tumour samples were collected through CONTICANET and stored in a centralised tumour data bank at the Bergonié Institute in Bordeaux, France, after obtaining approval from the appropriate ethics committees. This European fibromatosis tumour database contained exhaustive clinical information and follow-up data for over 5 years. From this tumour database, 187 eligible fibromatosis tumour tissues collected between December 1987 and January 2007 were tested in this retrospective study. Most of the patients with extra-abdominal fibromatosis had been surgically treated with curative intent. The tumour material comprised tumour samples from biopsies or microbiopsies performed using 16- or 18-gauge needles. The tissues had been obtained after surgery at diagnosis and snap-frozen in liquid nitrogen. Twenty-three formalin-fixed, paraffin-embedded (FFPE) tumour samples paired to frozen tumour samples were also obtained, as were 35-μm tumour slices, nucleic acids were obtained using Qiagen column separation according to the manufacturer's instructions . Available DNA for sequencing was obtained in 155 desmoid-type fibromatosis tumour biopsy specimens including all clinical forms of the disease. DNA lymphocytes from 96 anonymous individuals were used as non-mutated controls. The CTNNB1 mutations located in exon 3 were analysed by direct DNA sequencing. The genomic DNA was first amplified by polymerase chain reaction (PCR) of over 35 cycles at an annealing temperature of 55°C (forward primers: 5′-ATTTGATGGAGTTGGACATGGC-3′ and reverse: 5′-CCAGCTACTTGTTCTTGAGTGAAGG-3′) as previously described  was used.Following lysis of the 35 Conticanet series of tumours used for this study. Of 155 fibromatosis tissues from all topologies available for DNA sequencing, 101 extra-abdominal fibromatoses were used to investigate the correlation between β-catenin mutational status and clinical characteristics: age, sex, tumour size, tumour location , therapy )  were all represented in the β-catenin genotype, and the relationship between clinical characteristics and β-catenin mutational status was analysed using logistic regression analysis.Survival times were calculated from the date of diagnosis to the date of relapse . Log-rank tests were used for survival outcome analysis. Descriptive statistics were used to correlate patient characteristics to the β-catenin exon 3. No mutations were detected in the DNA extracted from lymphocytes of the 96 controls. All sequences were controlled in an independent experiment. The rate of mutation identified (83%) confirms and updates the findings of a preliminary report by our group on the basis of 95 cases from the same series  contained a mutation of β-Catenin missense mutations were essentially found at codons 41 and 45, identified as c.121 A>G or pThr-41-Ala (T41A), c.133T>C or pSer45Pro (S45P), and c.134C>T or pSer45Phe (S45F). The 41A and S45F mutations were relatively common, occurring in 41% (64 out of 155) and 34% (53 out of 155) of cases, whereas the S45P mutation occurred in just 6% (9 out of 155) ( of 155) . In threβ-catenin mutational status in daily practice.The clinical potential of the above results obtained from frozen tumour tissues led us to test the feasibility of sequencing using FFPE tissues, which are more readily available in daily practice. We therefore compared sequencing results from the FFPE samples paired to the frozen tumour specimens of the series. We observed identical data in 20 out of 23 (87%) samples, with no false positives and just three false negatives from FFPE tissues. Archival FFPE tumour tissues might thus provide useful and easily obtainable material for determining β-catenin mutation rate detected in this group (87% (88 out of 101)) was similar to that detected for the whole series, as was the distribution of mutation types , S45F, 36.5% (37 out of 101), and S45P, 9% (9 out of 101))  or R1 (34%). Few patients had received postoperative radiation therapy (18%) or medical treatment (8%). With a median follow-up of 62 months (range 3–452), 51 patients (51%) relapsed. β-Catenin mutational status was not found to be significantly associated with sex, age, tumour site, presentation (primary or relapse), or therapy. No correlation could be established between surgical resection quality and mutation rate.The European fibromatosis tumour database contained exhaustive clinical information, with follow-up data for over 5 years, for 101 patients (36 men) with extra-abdominal fibromatosis that had been surgically treated with curative intent. Fifty-seven patients had been treated for primary fibromatosis (57%) and 40 for relapse (40%) . The β-cof 101)) . We alsoβ-catenin mutational status correlated with clinical outcome in patients with extra-abdominal fibromatosis, we compared the frequency of disease-free survival (estimated from the date of diagnosis to the date of relapse) in patients with wild-type and β-catenin-mutated tumours. Among 51 relapses (51% of the cohort), 1 and 50 were from patients with wild-type tumour and mutated tumour, respectively. Five-year recurrence-free survival (β-catenin-mutated tumours (43%), compared with wild-type tumours (75%) (P=0.02). We then attempted to relate poor outcome to a specific genotype but found no significant differences for T41A, S45F, or S45P mutants, although the 45F mutation did seem to show a weak trend of significance for poorer outcome. Given the possible prognostic impact of surgical margin quality, we analysed the β-catenin genotype in patients with R0 resection (n=42) and found relapse to be significantly higher in patients with mutated tumours (18 out of 33) than wild-type tumours (0 out of 9) (P=0.02), as shown in the Kaplan–Meier graph , and in extra-abdominal forms in particular (n=101), results similar to that reported by another study (n=138). Remarkably, all mutations clustered at Threonine 41 (T41A) and Serine 45 (S45F and S45P) in all topographies, suggesting that these residues have crucial functions in the β-catenin/Wnt signalling pathway required for the maintenance of fibroblast/myofibroblast homoeostasis (P=0.02). However, no specific mutant genotypes  were related to prognosis, although we did observe a weak trend for S45F, but it was far from significant. Our results contrast with those of a very recent American study proposing that patients with fibromatosis harbouring an S45F mutation are at particular risk of recurrence  to cytoplasmic effectors and finally to the target gene – may contribute to the tumour phenotype. Second, alteration of the non-canonical Wnt pathway and effectors must also be considered. Hence, additional prospective studies are needed to find more discriminant markers.Evidence that β-catenin mutational status can be assessed using either fresh or archival extra-abdominal tumour specimens in accordance with the tumour collection system used in each pathology laboratory. This simple yet efficient molecular test might prove to be a very interesting complementary diagnostic tool to regular histological examination in extra-abdominal fibromatosis. Indeed, in cases in which there is insufficient tumour material for histological analysis, β-catenin sequencing could be performed, as it requires just a few ng of gDNA. The test could also be helpful to resolve puzzling cases in which the histological interpretation of primary tumour tissue obtained by fine-needle aspiration is complicated.Finally, from a practical point of view, the feasibility test comparing sequences from frozen and FFPE tumour tissues showed that A final application would be to use molecular tumour findings to individualise the choice of management protocols. This application of personalised medicine in patients with extra-abdominal fibromatosis could help to identify the right treatment at the right time for optimal outcome.


Interpretation of IOU in terms of vascular involvement by the tumour (ascompared to angiography or operative findings) was correct in 21 patients; no vascular invasionin 20 and portal vein invasion in the remainder. One false negative result occurred in a patientwhose IOU failed to show right hepatic artery encasement by the tumour. When compared topostoperative cholangiography or surgical specimen, IOU correctly demon-strated location andextent of the tumours in all but one patient who had incomplete tumour resection. IOU was alsohelpful in locating segment III duct for biliary bypass. The mean time used for IOU was 15.1 min(range 10-20 min.), and there was no procedure-related com-plication. When supplemented withoperative exploration, IOU seems to be very useful in the assessment of the resectability of hilarcholangiocarcinoma.The role of intraoperative ultrasonography (IOU) in the surgical treatment of hilar cholangiocarcinomawas explored in twenty-two patients, 17 males and 5 females. The mean age was55 years (range 36-78 years). Preoperative imaging studies included abdominal ultra-sonographyand/or CT scan, and visceral angiography. Operations performed were segment IIIbypass in 18 patients, local resection of tumour in 2 and resection of tumour


A squamous cell carcinoma of spontaneous orgin in a WHT/Ht mouse was used to study the frequency with which the regional axillary lymph nodes draining subcutaneous or intradermal tumours gave rise to tumours after their isogeneic transplantation as whole nodes. This frequency (similar to 40%) was found not to vary significantly with the size or duration of the tumour drained and not to be increased by coincident infective, traumatic or antigenic stimuli acting at the tumour site or in adjacent tissue. Because tumour growth occurred in only 2/55 (4%) nodes which were left in situ in mice whose tumours were radically excised, it was concluded that tumour forming node transplants reflected a small and limited content (estimated to be about 13) of transnodally passing tumour cells destined to pass on to the blood; separate experiments showed that tumour cells reaching the blood survived for only a few hours. Nodes from tumour-excised mice gave rise to tumours as frequently when autografted as when isografted to mice with no previous expose to the tumour. A review of the finding reported here and of previous quantitative data for this system enabled us to exclude any implication of anti-tumour immunity from our interpretation of the results of the experiments.


Tumours derived from these cells grew more rapidly in vivo than cells with normal dimethylarginine dimethylaminohydrolase I expression. Immunohistochemical and magnetic resonance imaging measurements were consistent with increased tumour vascular development. Furthermore, dimethylarginine dimethylaminohydrolase activity was detected in a series of human tumours. This data demonstrates that dimethylarginine dimethylaminohydrolase plays a pivotal role in tumour growth and the development of the tumour vasculature by regulating the concentration of nitric oxide and altering vascular endothelial cell growth factor production.Angiogenesis is a prerequisite for tumour progression and is highly regulated by growth factors and cytokines a number of which also stimulate the production of nitric oxide. Asymmetric dimethylarginine is an endogenous inhibitor of nitric oxide synthesis. Asymmetric dimethylarginine is metabolised by dimethylarginine dimethylaminohydrolase. To study the effect of dimethylarginine dimethylaminohydrolase on tumour growth and vascular development, the rat C6 glioma cell line was manipulated to overexpress the rat gene for dimethylarginine dimethylaminohydrolase I. Enhanced expression of dimethylarginine dimethylaminohydrolase I increased nitric oxide synthesis (as indicated by a two-fold increase in the production of cGMP), expression and secretion of vascular endothelial cell growth factor, and induced angiogenesis British Journal of Cancer (2002) 87, 673–680. doi:10.1038/sj.bjc.6600518www.bjcancer.comCancer Research UK© 2002  Angiogenesis is the formation of new capillaries from existing blood vessels. It is a multistep process that involves dissociation of endothelial cells (EC) from adjacent pericytes, remodelling of the extracellular matrix, proliferation and migration of EC and capillary differentiation. Insight into the regulation of this process is central to our understanding of a number of biological processes whether physiological, as occurs during development, the reproductive cycle and wound healing, or pathological, as occurs during atherogenesis, diabetic retinopathy and tumour progression .There is strong evidence to suggest that nitric oxide (NO), produced from arginine by the nitric oxide synthases (NOS), is a crucial signalling molecule and regulator of angiogenesis . NO enhaL-NMMA) are competitive inhibitors of NO synthesis. Free ADMA is found in plasma and urine of healthy individuals and is synthesised by the post-translational methylation of protein arginine residues and liberated upon their hydrolysis  and N-mono methylarginine  foetal calf serum. The full length rat DDAH I cDNA was generated by RT–PCR using mRNA isolated from C6 cells, forward primer: GGAGCAAGCTTCGCCACCATGGCCGGCCTCAGCCA and reverse primer: GCCTGCTCGAGTCAAGAGTCTGTCTTCTTGTTAAT and subsequently cloned into HindIII/XhoI sites in pcDNA 3.1/hygro (InVitrogen). C6 cells were transfected using the poly L-ornithine method  and selected in medium supplemented with 500 μg ml−1 hygromycin B  were maintained in Ham's F-10 medium containing 5 m6 cells in 0.1 ml PBS. Tumour growth was measured at daily intervals after anaesthetising with fluothane in 2% N2O, 5% O2. The tumour volume was calculated using the formula: tumour volume=π/6·(length×width×depth). All procedures were performed in accordance to the Home Office Scientific Procedures Act 1986.Female (7–8 weeks old) MF1/nu mice were injected subcutaneously in the flanks with 2×10The protein concentration of cell lysates and tumour homogenates were determined by Bradford assay and equal amounts analysed by SDS–PAGE. DDAH expression was detected using a monoclonal antibody against rat DDAH I  for 1 h at 37°C. Cells were washed twice in ice-cold Krebs solution and lysed in 0.1% (w v−1) SDS. Human tumour biopsy specimens taken during surgical removal of the tumour, or mouse tumour tissues were homogenised in 10 mM sodium phosphate buffer pH 6.6 using a motor homogeniser. Aliquots of 500 μl were incubated with [14C] L-NMMA (0.4 μCi ml−1) for 1 h at 37°C. The formation of [14C] citrulline was determined as previously described . The high identity in the cDNA sequences of human and rat or mouse VEGF ensures the crossreactivity of the probe . The probe for VEGF was a 540 bp −1). Tumours were excised at an early stage of development (day 15), weighed, cut in small pieces and maintained in culture medium for 4 days. VEGF secretion was measured in the culture medium. Values were correlated with the protein concentration of cells or the weight of tumours, respectively.Culture medium was collected from confluent 6-well plates and analysed for VEGF expression using a murine VEGF ELISA kit . Mice bearing tumours derived from D27  or C6  wild type cells were anaesthetised with a single intraperitonal dose of 10 ml kg−1 Hypnorm/Hypnovel and placed so the tumour hung into the centre of the surface coil. The mouse core temperature was maintained at 37°C by a warm water blanket. Multi-gradient echo (MGRE) images were acquired from three transverse 1 mm thick slices through the centre of the tumour. The readout gradient was oscillated to generate a series of eight echoes, utilising a repetition time of 100 ms, initial echo of 5 ms and an echo spacing of 5 ms, resulting in sets of images with decreased R2* weighting. Tumour R2* maps for each slice were generated using all eight gradient-echo image sets by fitting an exponential model on a pixel-by-pixel basis (2* was determined from a region-of-interest (ROI) encompassing the whole tumour but excluding the surrounding skin/muscle.A separate cohort of size-matched D27 and C6 wild type tumours were imaged at 18 and 23 days post-passage respectively. −1 of the perfusion marker Hoechst 33342 (Sigma) 1 min prior to cervical dislocation. Tumours were rapidly excised, frozen and sections cut (10 μm). Acetone fixed sections were visualised by fluorescence microscopy. Fluorescence signals from entire tumour sections were recorded with digital images acquired under identical conditions. The area of tumour stained by Hoechst 33342 as a percentage of the total area of tumour section was calculated using anaLYSIS .Mice from the same cohort as used for the MRI and bearing similarly size-matched tumours were injected intravenously with 15 mg kgLocal Ethical Committee approval was obtained for the collection of human tissues.14C radiolabelled L-NMMA to L-citrulline  and growth  that constitutively express DDAH I and mock transfected clones  were analysed. Western blot analysis of D20, D26 and D27 cells revealed similarly high levels of DDAH I protein expression  compared to the wild type or the control M4 cells  MR images allow the relaxation rate R2* to be quantified, which is directly proportional to the tissue content of deoxyhaemoglobin and hence a sensitive index of tissue vascularity  indicates a higher concentration of deoxyhaemoglobin and increased tumour vascularisation  was employed to assess the blood vessel development  tumours .n=4).The effect of DDAH I on tumour perfusion was examined using the fluorescent marker Hoechst 33342  beads and embedded in fibrin gels were incubated with conditioned medium from confluent cultures of C6, D27 or SGHEC-7 cells and the formation of invasive processes was analysed over a period of 2 days. Invasion of endothelial cells into the fibrin gel was used as an indicator of the angiogenic potential of factors produced by the D27 cells. The average of the maximum length of the process minus the radius of the bead from 40 MC beads was determined. Statistical analysis showed that conditioned medium from D27 cells significantly stimulated the migration of SGHEC-7 cells into the fibrin gel compared to medium from C6 or SGHEC-7 cells . Therefore, a possible role of DDAH I in vessel development could be the facilitation of the endothelial cell invasion. In support of this, conditioned medium from D27 cells significantly stimulated the migration of SGHEC-7 cells into the fibrin gel compared to medium from C6 or SGHEC-7 cells. These results demonstrate that DDAH activity and the intracellular concentration of ADMA regulate the release of an angiogenic factor or factors in vivo .Vessel growth and development is under the control of a number of factors the most potent being VEGF. VEGF is a largely endothelial cell-specific mitogen that is induced by hypoxia. It promotes angiogenesis, blood vessel permeability and maintains tumour vessel integrity (Our findings indicate that human tumours exhibit DDAH activity. Although this study is limited in number it does suggest that human brain tumours express particularly high levels of DDAH activity. Increased NO production has also been reported in human CNS tumours (In summary, this study shows that increased DDAH expression results in decreased tumour ADMA, increased NO and VEGF production, vascularisation and tumour growth. Our results provide the first demonstration of the importance of the ADMA/DDAH pathway in the regulation of vessel development. It also has important implications for the regulation of angiogenesis in other pathological and physiological circumstances in which VEGF expression or NO production is involved. The fact that tumour development is so dramatically altered by regulating this pathway adds a novel level of complexity to a system where the precise balance between pro- and anti-angiogenic factors is of key importance. Therapeutic interventions in oncology using the DDAH/ADMA pathway are therefore plausible.


An apparatus which has been widely used in rats for measuring swelling of the foot induced locally by inflammatory agents has been adapted to measure rapidly, accurately and objectively, the growth of tumour cells transplanted to the foot, and the reactions of the normal tissues (tumour bed) to tumour growth. General features on the apparatus and the techniques used are described. Examples are provided of preliminary measurements made of normal growth of the foot, reactions of the foot to two injurious agents (histamine and Corynebacterium parvum) and of growth of allogeneic (W256) tumour cells.


Human gastric carcinomas were tested for their immunohistochemical reactivity with anti-type I procollagen antiserum. In all specimens of scirrhous carcinomas, staining of the tumour cells was strongly positive, while in medullary carcinomas staining of the tumour cells was generally poor. These results suggest that the tumour cells in scirrhous carcinomas produce collagen in their stroma.


The chemosensitising effects of poly(ethylene oxide)-poly(propylene oxide)-poly-(ethylene oxide) (PEO-PPO-PEO) block copolymers (Pluronic) in multidrug-resistant cancer cells has been described recently . This paper presents initial studies on in vivo evaluation of Pluronic copolymers in the treatment of cancer. The anti-tumour activity of epirubicin (EPI) and doxorubicin (DOX), solubilised in micelles of Pluronic L61, P85 and F108, was investigated using murine leukaemia P388 and daunorubicin-sensitive Sp2/0 and -resistant Sp2/0(DNR) myeloma cells grown subcutaneously (s.c.). The study revealed that the lifespan of the animals and inhibition of tumour growth were considerably increased in mice treated with drug/copolymer compositions compared with animals treated with the free drugs. The anti-tumour activity of the drug/copolymer compositions depends on the concentration of the copolymer and its hydrophobicity, as determined by the ratio of the lengths of hydrophilic PEO and hydrophobic PPO segments. The data suggest that higher activity is associated with more hydrophobic copolymers. In particular, a significant increase in lifespan (T/C> 150%) and tumour growth inhibition (> 90%) was observed in animals with Sp2/0 tumours with EPI/P85 and DOX/L61 compositions. The effective doses of these compositions caused inhibition of Sp2/0 tumour growth and complete disappearance of tumour in 33-50% of animals. Future studies will focus on the evaluation of the activity of Pluronic-based compositions against human drug-resistant tumours.


Recently, cytokine gene transfer into tumour cells has been shown to mediate tumour regression in animal models via immunomodulation. Consequently, a number of clinical protocols have been developed to treat cancer patients with cytokine gene-modified tumour cells. Here, we report the results of a clinical phase I trial using for the first time autologous, interleukin 7 gene-modified tumour cells for vaccination of ten patients with disseminated malignant melanoma. Melanoma cells were expanded in vitro from surgically removed metastases, transduced by a ballistic gene transfer technique and were then injected after in vitro irradiation s.c. at weekly intervals. Clinically, there was no major toxicity except for mild fever, and no major clinical response towards vaccination was observed. Eight of ten patients completed the initial three s.c. vaccinations and were eligible for immunological evaluation. Post vaccination, peripheral mononuclear cells (PBMCs) were found to contain an increased number of tumour-reactive proliferative as well as cytolytic cells, as determined by a limiting dilution analysis. In three of six patients, the frequencies of anti-melanoma cytolytic precursor cells increased between 2.6- and 28-fold. Two of these patients showed a minor clinical response. Analysis of the autologous tumour cell vaccines regarding IL-7 secretion after gene transfer, HLA class I and class II cell surface expression, secretion of immunosuppressive mediators  and various melanoma-associated tumour antigens revealed a very diverse expression profile. In conclusion, vaccination using gene-modified autologous melanoma cells induced immunological changes in a group of advanced, terminally ill patients. These changes can be interpreted as an increased anti-tumour immune response. However, immunological modulation was most pronounced in patients in good physical condition. Therefore, patients with minimal tumour load or minimal residual disease might preferentially benefit from tumour cell vaccination in further studies. In order to evaluate the effects of the cytokine gene-modified tumour cell vaccines more precisely, an antigenically better defined vaccine is needed.


Eighteen patients with inoperable hepatocellular carcinoma (HCC) were treated with intrahepatic arterial yttrium-90 microspheres. All these patients showed a lung shunting below 15% and a tumour-to-normal ratio higher than 2 as determined by diagnostic technetium-99m macroaggregated albumin (Tc-MAA) gamma scintigraphy. The treatment was given through an arterial port placed during laparotomy. The radiation doses to the liver and tumour were determined intraoperatively with a beta probe and liquid scintillation counting of multiple liver biopsies. The treatment was well tolerated without major complications. In all patients the tumour marker fell to a level which ranged from 41% to 0.2% of the pretreatment level. Tumour regression was found to be dose related. Progressive or static disease occurred in a higher proportion of patients whose tumours received < 120 Gy (P = 0.005). Survival was better in those whose tumours received > 120 Gy  than those whose tumours received lower doses . This difference is statistically significant with P = 0.005. We conclude that yttrium-90 microsphere therapy is safe and that tumour response is dose related. A tumour dose of > 120 Gy is recommended.


Tumour mass, volume and the presence or absence of peritoneal carcinomatosis were determined at sacrifice on day 38. Tumours from each group were stained for markers of blood vessels, proliferation and apoptosis. To further define the time frame of the vascular-targeting effects of chronic therapy with ZD6126, TMK-1 cells were again injected into the gastric wall of mice in a second experiment. On day 14, a single i.p. injection of ZD6126 100 mg kg−1 mouse−1 or vehicle was delivered. Groups of three mice each were killed and the tumours harvested at days 1, 3 and 5 post-ZD6126 injection. Tumours were processed and stained for endothelial and tumour cell apoptosis and proliferation. No overt toxicity was observed with ZD6126 therapy. ZD6126 led to a marked inhibition of tumour growth (82% decrease vs control (P<0.001)). ZD6126 also led to a significant decrease in the incidence of peritoneal carcinomatosis  (P<0.01). Histological analysis of tumours revealed large regions of central necrosis in the treated group, as well as a dramatic increase in tumour cell apoptosis (7.4-fold increase (P<0.001)), consistent with the vascular-targeting activity of ZD6126. Mice treated with ZD6126 demonstrated a 59% decrease in PCNA-positive cells (P< 0.02), indicating reduced tumour cell proliferation. In addition, tumours treated with ZD6126 exhibited a 40% decrease in microvessel density (P<0.05). Results from mice treated with a single injection of ZD6126 demonstrated the acute effects this agent has on the tumour vasculature. The ratio of endothelial cell apoptosis to endothelial cell proliferation was increased within 24 h of a single injection. In conclusion, ZD6126 significantly inhibited tumour growth and metastasis in an orthotopic model of human gastric adenocarcinoma, without detectable problematic adverse effects. These data suggest that ZD6126 may be worthy of investigation in the treatment of primary gastric adenocarcinoma.The purpose of this study was to examine the effects of ZD6126, a novel vascular-targeting agent, on tumour growth and angiogenesis in an orthotopic model of gastric cancer. TMK-1 human gastric adenocarcinoma cells were injected into the gastric wall of nude mice. After the tumours were established (day 14), therapy was initiated. Mice ( The 5-year survival rate for patients with gastric cancer in the United States is only 5–15%  and in vivo vascular responses  have been described that could play a significant role in their anti-tumour effects , and was cultured and maintained in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, as described previously . ZD6126 was dissolved in 5% sodium carbonate and diluted to the final volume with phosphate-buffered saline (PBS), as previously described , were acclimated for 1 week and caged in groups of five. All animal studies were conducted under the guidelines approved by the Animal Care and Use Committee of The University of Texas MD Anderson Cancer Center, and met all of the standards required by the UKCCCR guidelines for the welfare of animals in experimental neoplasia, as published (−1) and, under sterile conditions, subjected to an upper midline laparotomy. TMK-1 cells (106) in Hank's balanced salt solution were injected into the wall of the mid-stomach. After 14 days, when tumours were approximately 1–2 mm in diameter, mice were randomly assigned to one of three groups: (a) daily-dose control ; (b) weekly-dose ZD6126 ; and (c) daily-dose ZD6126  , indicating that they would be expected shortly to become moribund. Body weights were measured, tumours were excised, tumour weight and diameters were subsequently determined and the presence or absence of peritoneal carcinomatosis was noted. Tumour volumes were calculated by the equation width2 × length × 0.5. Tumour tissue was harvested and placed in either 10% buffered formalin for paraffin fixation or optimal cutting temperature (OCT) compound  and frozen in liquid nitrogen for subsequent immunohistochemical analysis.Mice were anaesthetised by i.p. injection of sodium phenobarbital  or control solution (PBS+5% sodium carbonate) 14 days after tumour-cell implantation. These mice were injected i.p. with 1 mg/0.2 ml of 5-bromo-2′-deoxyuridine (BrdU)  dissolved in PBS 1 h before euthanasia to determine the fraction of proliferating cells. Three mice per group were euthanised at days 1, 3, or 5 after a single injection of ZD6126, and the tumour tissues were harvested as above.In a separate experiment to clarify the timing of the antivascular effect of ZD6126, mice bearing established orthotopic tumours were injected with a single dose of ZD6126 . The antibodies used were as follows: rat anti-mouse CD31 , mouse anti-BrdU , mouse anti-human PCNA PC-10 , DeadEnd Fluorometric TUNEL system , rat anti-mouse IgG2a , Texas Red goat anti-rat secondary antibody  and goat anti-mouse IgG Alexa Fluor 488 .Tissue sections were sectioned and stained for H&E, CD31 (vessels), TUNEL (apoptotic cells), BrdU (proliferative cells) and PCNA (proliferative cells), as previously described  were avoided. The ratio of endothelial cell apoptosis to endothelial cell proliferation was determined by counting the number of CD31-positive/TUNEL-positive cells divided by the number of CD31-positive/BrdU-positive cells per high-power field.Sections were examined using a Zeiss photomicroscope  equipped with a 3-chip charge-coupled device colour camera . The images were analysed using Optimas image analysis software . Positive cells were counted using Scion software, based on the NIH Image program for Macintosh . Quantification of positive cells was expressed as the average of the number of cells in 0.05-mmt-test, the χ2 test, or, for analysis of nonparametric data, the Mann–Whitney U-test with InStat Statistical Software . The results of the in vivo experiments were tested for outliers using Grubb's test (www.graphpad.com). A P-value of less than 0.05 was considered statistically significant.Statistical differences among groups were examined using the two-tailed Student's P< 0.05) and the daily-dose group  , but weekly ZD6126 did not significantly reduce tumour mass relative to that in the control group (P=0.26).No difference was discerned in the body weight or grooming habits of the treatment groups . The effects of ZD6126 on the growth of TMK-1 cells implanted into the stomach walls of nude mice are shown in P<0.01) (P=0.08 vs controls).TMK-1 cells are known to induce peritoneal carcinomatosis in a similar mouse model (P<0.01) . Weekly P<0.05)  and 58% fewer in the daily group (P<0.05)), concomitant with significant increases in TUNEL-positive cells in both groups (5.3-fold increase in the weekly group (P<0.007) and 7.4-fold increase in the daily group (P<0.001)).The proliferative and apoptotic rates of the tumour cells in each of the three groups are shown in P<0.004)). There was also a concomitant increase in cell death at day 1, which was sustained through to day 5 (15-fold increase at day 1 (P<0.05) and nine-fold increase at day 5 (P<0.05)). Importantly, there was also a significant decrease in endothelial cell proliferation by day 1 post-ZD6126 injection (56% decrease in CD31+/BrdU+ cells (P<0.05)). However, since the process of angiogenesis is determined by a balance between proliferating and apoptotic endothelial cells, the ratio of these two parameters was determined. As shown in P<0.01). This increase was apparent as early as 24 h post-ZD6126 injection, and continued through day 5.Tumours harvested from mice treated with ZD6126 either weekly or daily for 38 days were stained for markers of endothelial-cell apoptosis (CD31/TUNEL), to determine whether the decrease in microvessel density was associated with increased endothelial cell apoptosis. However, at that relatively late time point, no apoptotic endothelial cells were detected (data not shown). Therefore, to examine the acute effects of ZD6126 on the tumour microvasculature, mice bearing established tumours were injected with a single dose of ZD6126 (100 mg/kg) on day 14, and tumours were harvested 1, 3, or 5 days after the injection. As shown in Our findings in this study demonstrate the efficacy of selectively targeting the tumour vasculature with the novel antivascular agent ZD6126 in an orthotopic model of human gastric cancer. Previous studies have described ZD6126, both as a single agent and in combination with radiation and chemotherapy, in various models in which tumour cells are implanted in subcutaneous tissues . Here we−1, 5 days per week) caused a significant inhibition of tumour growth. Once-weekly dosing, in contrast, reduced the tumour volume, but did not significantly reduce tumour mass. This difference may be due to the variability of excising and weighing the tumour, as it is difficult to ensure complete removal of the tumour, without the accompanying uninvolved stomach wall. This is reflected by the proportionately greater variability in the tumour mass measurement, especially as the tumour volume is reduced. Previous studies in subcutaneous xenograft tumour models have shown that a single bolus dose of ZD6126 produces widespread central tumour necrosis with a rim of viable tumour cells surviving treatment, which can quickly repopulate the tumour mass, resulting in limited tumour growth delay (Daily dosing with ZD6126 (100 mg kgExamination of the tumour samples at the end of the experiment (day 38), 24 h after the last dose of ZD6126 was administered, showed that both treatment schedules produced significant antitumour effects in terms of increased tumour cell death (TUNEL-positive staining), together with decreased tumour cell proliferation (PCNA-positive staining). However, the TUNEL-staining procedure can detect both apoptotic and necrotic cell death , probably as a result of drug-induced effects on endothelial cell shape, leading to exposure of basement membrane and rapid induction of endothelial cell apoptosis and subsequent loss of vessel function  significantly inhibited tumour growth in both the subcutaneous and orthotopic settings  or human (SW620) colorectal cancer (The present study has shown that the vascular-targeting agent ZD6126 was effective in reducing the growth and peritoneal spread of human gastric cancer cells implanted orthotopically into the stomach walls of nude mice. Furthermore, the antitumour activity was improved with daily dosing compared with weekly dosing. Further studies are needed to more completely define the mechanism of action of this agent and to determine whether additional antitumour effects can be seen in this model in combination with clinically relevant chemotherapy, radiation therapy, or antiangiogenic agents. However, the present preclinical results provide support for investigating the potential activity of ZD6126 in gastric cancer in the clinic.


The aim of this study was to compare interstitial laser thermotherapy with excision of a liver tumour. A dimethylhydrazine-induced adenocarcinoma was transplanted (implanted if not stated otherwise) into the left lateral lobe of the rat liver, and treatment was performed 8 days later. In the main experiment, rats were treated with resection of the tumour-bearing lobe or underwent interstitial laser thermotherapy, which was performed at a steady-state temperature of 46 degrees C for 30 min, 3 mm from the tumour margin. The incidence and extent of intraperitoneal spread was smaller after laser thermotherapy than after resection of the tumour-bearing lobe, with no difference in local control. Metastatic spread after resection of the median liver lobe was similar to that observed after sham procedures for thermotherapy or resection, suggesting that the advantage of thermotherapy was not due to a difference in surgical trauma. Additional studies showed that laser thermotherapy reduced intraperitoneal spread when treatment was suboptimal or in a tumour inoculation model and suggested that immunological mechanisms might be involved. It is concluded that interstitial laser thermotherapy reduces spread of liver tumour compared with resection.


We report a rare case of a primary leiomyosarcoma of the adrenal gland. A 73-year-old woman presented with an inferiorvena cava syndrome. MR imaging was suggestive of a large tumour originating from the right adrenal gland. Angiographyrevealed a tumour vascularised by the right adrenal artery. At explorative laparotomy a tumour of 27 cm in diameter wasfound which was completely fixed to the liver; the tumour was therefore considered unresectable. As a consequence of themechanical problems caused by this large tumour, the patient died 3 weeks after the operation. Autopsy revealed no distantmetastases or other primary tumour site.


Genetic studies have been made in two cases of primary choriocarcinoma from patients in whom the antecedent pregnancy was a hydatidiform mole. Restriction fragment length polymorphisms of the DNA from the tumour, the patient and her partner were examined and in both cases the tumours were shown to be androgenetic in origin, having only paternal polymorphisms. While one tumour was shown to be heterozygous, two different paternal alleles being demonstrated with some probes, the other tumour was shown to be homozygous for all informative polymorphisms examined. Thus choriocarcinoma can follow complete hydatidiform mole which may be either heterozygous or homozygous.


Min mouse model of colon cancer, if selective COX-2 and MMP inhibitors provide additive or synergistic therapeutic benefits in intestinal tumour prevention. The broad-spectrum MMP inhibitor  and the selective COX-2 inhibitor  both showed dose-dependent inhibition of the number of adenomas in Min mice. Using suboptimal doses, the MMPI reduced tumour multiplicity by 32%, the COX-2I by 48% and, both agents in combination resulted in a 67% decrease compared to control demonstrating a cooperative effect on intestinal tumorigenesis. Apoptosis, proliferation, and angiogenesis were assayed in tumors from each treatment group. These agents in combination allowed for a lowered dosage to be administered to achieve significant biological effects. Clinically, this could potentially reduce side effects associated with currently used MMP and COX-2 inhibitors. Together, these compounds could represent an easily tolerated chemopreventive approach.Matrix metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2) are expressed in both sporadic and familial adenomatous colonic polyps and tumours and have been independently shown to play causal roles in intestinal tumour formation in mouse models of colon cancer. The apparent roles of these enzymes in intestinal tumorigenesis led us to examine, in the  Colon cancer is the third largest cause of cancer-related deaths in both men and women in the US. Sporadic colorectal cancer is a multistep process involving a number of genetic and epigenetic changes , while fInhibitors of cyclooxygenase , the enzyme that catalyses the conversion of arachidonic acid to prostaglandin, have been utilised as chemopreventive agents for colorectal cancer. There are two forms of the COX enzyme; COX-1 is expressed ubiquitously while COX-2 expression is induced in response to a number of different stimuli including cytokines, growth factors, oncogenes, serum, and tumour promoters (Min) mouse  family are induced in colon cancer , was decreased by 48% as compared to vehicle-treated mice (Min adenomas express MMP-2 and MMP-10 (stromelysin-2) and 50% express MMP-3 and MMP-13 (collagenase) with expression limited to the stroma surrounding the adenoma  and a selective COX-2 inhibitor (A-285969) to Min mice significantly suppresses intestinal tumorigenesis, and that administration of both the MMP inhibitor (MMPI) and the COX-2 inhibitor (COX-2I) in combination further reduces intestinal tumorigenesis to a level that is greater than either compound individually.Since COX-2 and MMP-7 are not coregulated and are unlikely to be part of the same signal-transduction pathway, we hypothesised that combination therapy with selective COX-2 and MMP inhibitors may provide additive or synergistic therapeutic benefits in the Min (Min) mice  were maintained as previously described with littermates housed under identical conditions in microisolator cages . Analysis of the Min allele was performed as described using a PCR assay  suspended in 0.2% hydroxypropylmethylcellulose (HPMC), A-285969 at 40 mg kg−1  suspended in HPMC. Sulindac was given as a positive control for tumour inhibition and was given at a dose of 1.875 mg kg−1  while HPMC-injected mice were used as control (n=3). Combination study: mice were administered A-177430 at a dose of 30 mg kg−1  suspended in 0.2% HPMC, A-285969 at 20 mg kg−1  suspended in HPMC, both A-177430 and A-285969 at the doses used in single compound treatment (n=6), or vehicle (HPMC) alone (n=5). All compounds were administered by intraperitoneal (i.p.) injection twice daily for 45 days beginning at 5 weeks of age. The twice daily dose of 40 and 20 mg kg−1 of A-285969 was chosen because pilot studies have shown A-285969 to be active in a mouse cornea model of angiogenesis at 50 mg kg−1  and because doses of 20 mg kg−1 have shown to be safe on the gastrointestinal tract of dogs (Bell R in preparation). The twice daily dose of 100 and 30 mg kg−1 of A-177430 was chosen because these doses have been previously shown to be effective in inhibiting tumour growth of the MatLyLu prostate cancer cell line in rats  in 1 × PBS and transferred to 70% ethanol for storage at 4°C. Intestines were cut longitudinally and tumours were analysed under a dissecting microscope at × 10 magnification. Tumour sample identifications were masked and the number, diameter, and distribution of tumours were recorded.For analysis of intestinal tumours, animals were killed by COμm) were cut and mounted on Superfrost Plus slides . Slides were stained for apoptosis using the ApopTag Kit  according to the manufacturer's instructions. Nuclei were visualised by counterstaining with Contrast Green. Apoptosis results were quantified by counting the number of positively stained tumour-associated nuclei per 1000 total tumour-associated nuclei. Results are expressed as percent positively stained nuclei.Tumours were dissected out of the fixed intestines and embedded in paraffin. Sections . Antigen retrieval was performed with Target Retrieval solution  according to the manufacturer's instructions. Negative controls consisted of sections that were incubated in control IgG antibody. Endogenous peroxidase and biotin were quenched with 0.03% hydrogen peroxide and Biotin Blocking System , respectively. A primary antisera dilution of 1 : 50 was prepared using the Animal Research Kit/Peroxidase  biotinylation and blocking reagents and slides were incubated for 6 h. Diaminobenzidine (DAB) plus was used to produce localised, visible staining followed by a haematoxylin counterstain.Immunohistochemistry results were quantified by counting the number of positive tumour-associated nuclei out of the total number of tumour-associated nuclei with a minimum of 600 nuclei analysed for each tumour. Results are expressed as percent of nuclei staining positive.Analysis of angiogenesis was performed with a rabbit anti-human Von Willebrand factor (VWF) antibody obtained from DAKO Laboratories. Endogenous peroxidases were quenched with 0.6% hydrogen peroxide. Antigen retrieval was accomplished by proteinase K digestion in a solution of 4% proteinase K for 30 min at room temperature. Sections were incubated in control IgG antibody (negative control) or rabbit anti-VWF antibody (1 : 2000) overnight at 4°C and developed with the Vectastain ABC kit from Vector Laboratories  according to the manufacturer's instructions. Immunohistochemistry staining for VWF was quantified using Zeiss Image  and results are expressed as percent of total tumour area stained.P-value is less than 0.05 as determined with the nonparametric Mann–Whitney test.All values were determined using the StatView program . A value was determined to be statistically significant if the 50 values of 2–6 nM for MMPs -1, -2, -3, -7, and -9. This MMPI has been used successfully at 100 mg kg−1 to inhibit tumour growth and metastasis in rats inoculated with MatLyLu rat prostate cancer cells , prostaglandin-driven inflammation (ED50=1.0 mg kg−1) in adjuvant arthritis in rats, and angiogenesis in a mouse cornea model of angiogenesis (50 mg kg−1) .The MMP inhibitor A-177430 (referred to as MMPI) is a previously described broad-spectrum MMP inhibitor , MMPI at 100 mg kg−1 (200 mg kg−1 day−1) or COX-2I at 40 mg kg−1 (80 mg kg−1 day−1) as indicated in Materials and Methods. The NSAID sulindac was used as a positive control. Both the MMPI and the COX-2I reduced tumour multiplicity by 69% (5.6±2.5) and 64% (6.4±1.5), respectively, compared to vehicle-treated controls (18±2). Sulindac was less effective than either of the MMPI or the COX-2I in this experiment  or suboptimal doses of MMPI at 30 mg kg−1 (60 mg kg−1 day−1), COX-2I at 20 mg kg−1 (40 mg kg−1 day−1) or both MMPI and COX-2I at the same doses used in the individual treatments as indicated in Materials and Methods. Tumour multiplicity was reduced in MMPI-treated mice by 32%  and by 48%  in COX-2I-treated mice as compared to vehicle-treated mice (13.2±2.8) as shown in P=0.0062) as compared with vehicle-treated mice. Additionally, the reduction in tumour number in combination-treated mice was significantly greater than mice treated with either compound individually . Similarly, tumours from mice treated with COX-2I exhibited an increased apoptotic index of four-fold as compared with vehicle-treated mice . Additionally, tumours from mice treated with the combination of the MMP and COX-2 inhibitors had a 2.5-fold increase in the apoptotic index  compared to tumours from mice treated with vehicle; however, there was no significant increase in the rate of apoptosis over treatment with either MMPI or COX-2I alone . Tumours from Min mice treated with the combination MMPI and COX-2I did not exhibit a significantly different rate of proliferation than tumours from mice treated with the COX-2I alone  or MMPI (P=0.0009) and COX-2I (P=0.0031) used individually  and a selective COX-2 inhibitor (A-285969) could cooperate to reduce intestinal tumour formation in Min tumour multiplicity in a dose-dependent manner. These results are consistent with previously published data using structurally distinct MMP and COX-2 inhibitors (Min mice with both MMP and COX-2 inhibitors resulted in an additive decrease in the average tumour number.Both the MMPI and the COX-2I decreased The mechanism of inhibition of tumour number was investigated by assaying for tumour apoptosis, proliferation, and angiogenesis. The MMPI alone raised the level of tumour cell apoptosis, but did not significantly affect proliferation or angiogenesis. An increase in apoptosis is apparently sufficient to reduce the number of detectable adenomas. The apoptotic activity of MMPs and of their endogenous inhibitors, TIMPs, is a controversial subject. In some cases, MMPs have been shown to be proapoptotic while in other cases they are implicated to be antiapoptotic (for review see The COX-2I also increased tumour cell apoptosis, consistent with previous reports of proapoptotic effects of COX-2 inhibition . Thus, tThe COX-2I reduced adenoma proliferation as determined by Ki67 staining. Several studies have shown prostaglandins to be important modulators of cell proliferation , and recThe most interesting observation of these studies is the effect of the MMPI and COX-2I on tumour-associated angiogenesis. Neither compound significantly altered angiogenesis alone at the doses given, but the combination resulted in a 53% reduction in VWF-stained blood vessels. Previous studies have indicated that both COX-2 and MMP inhibition have antiangiogenic effects . In prelMMP inhibitors have been successfully used in preclinical models to demonstrate that MMPs are important in several different tumour types .Min mice with NSAIDs and EGF receptor kinase inhibitors cooperate to inhibit tumour multiplicity. Additionally, the combination of an MMP inhibitor with a COX-2 inhibitor may be used with traditional cytotoxic agents, based on promising results observed with such agents used in combination with either a MMP or COX-2 inhibitor (Treatment with two different inhibitors with independent signalling pathways may decrease the toxicity of drugs by reducing the dose necessary to achieve the same effect. Recent findings (nhibitor . These d


Efficacy of cancer immunotherapy with cultured tumour-infiltrating lymphocytes (TILs) depends upon infused TILs migrating into tumour-bearing tissue, in which they mediate an anti-tumour response. For TILs to enter a tumour, they must first bind to tumour endothelium, and this process depends on TILs expressing and regulating the function of relevant cell-surface receptors. We analysed the cell-surface phenotype and endothelial binding of TILs cultured from human melanoma and compared them with peripheral blood T cells and with allostimulated T cells cultured under similar conditions. Compared with peripheral blood T cells, TILs expressed high levels of five integrins, two other adhesion molecules, including the skin homing molecule CLA, and several activation markers and showed markedly enhanced integrin-mediated adhesion to a dermal microvascular endothelial cell line in vitro. Compared with the allostimulated T cells, TILs expressed higher levels of the cutaneous lymphocyte antigen (CLA), the adhesion molecule CD31 and the activation markers CD30 and CD69, but lower levels of several other adhesion and activation molecules. These phenotypic and functional properties of TILs should have complex effects on their migration in vivo. Expression of CLA, the skin homing receptor, may increase migration to melanoma (a skin cancer), whereas integrin activation may cause non-specific binding of TILs to other endothelium. Manipulation of the culture conditions in which TILs are expanded might result in a phenotype that is more conducive to selective tumour homing in vivo.


MUC4 expression in vivo using clonal human pancreatic tumour cells (CD18/HPAF) grown either orthotopically in the pancreas (OT) or ectopically in subcutaneous tissue (SC) in the nude mice. Histological examination of the OT and SC tumours showed moderately differentiated and anaplastic morphology, respectively. The OT tumour cells showed metastases to distant lymph nodes and faster tumour growth (P<0.01) compared to the SC tumours. The MUC4 transcripts in OT tumours were very high compared to the undetectable levels in SC tumours. The SC tumour cells regained their ability to express MUC4 transcripts after in vitro culture. Immunohistochemical analysis using MUC4-specific polyclonal antiserum confirmed the results obtained by Northern blot analysis. Interestingly, the OT tumours showed expression of TGFβ2 compared to no expression in SC, suggesting a possible link between MUC4 and TGFβ2. The MUC4 expression, morphology, and metastasis of human pancreatic tumour cells are regulated by a local host microenvironment. TGFβ2 may serve as an interim regulator of this function.MUC4 is highly expressed in human pancreatic tumours and pancreatic tumour cell lines, but is minimally or not expressed in normal pancreas or chronic pancreatitis. Here, we investigated the aberrant regulation of  O-glycosidic linkage with oligosaccharides. These domains are composed of tandemly repeated sequences that vary in number, length, and amino-acid sequence from one mucin to another , gel forming , and soluble (MUC7) . The COOH-terminus of MUC4 encodes 12 distinct domains that include two cysteine-rich domains, three epidermal growth factor (EGF)-like domains, two regions rich in potential N-glycosylation sites, one hydrophobic transmembrane region, and one short cytoplasmic tail. Nine out of 12 of the C-terminal domains share 60–80% similarity in sequence with the rat sialomucin complex, known as SMC. Sialomucin complex is the rat homologue of human MUC4 and is now known as rat Muc4  sites of the nude mouse. The morphologically differentiated invasive OT tumours demonstrated a high level of expression of MUC4 mRNA and protein compared to undetectable levels in poorly differentiated SC tumours. However, the in vitro culture of SC tumour cells resulted in the expression of MUC4 transcripts comparable with its expression level in the parental cell line CD18/HPAF. Paracrine stimulation by growth factors and cytokines has been demonstrated to be one of the mechanisms responsible for the organ preference and proliferation of the tumour cells. The MUC4-expressing OT tumours also showed transforming growth factor (TGF)β2 expression. The study suggests that the site of pancreatic tumour growth in vivo strongly influences MUC4 and TGFβ2 expression, tumour morphology, and invasiveness of CD18/HPAF cells.In the present study, the influence of the local host microenvironment on the expression of the Female athymic mice (nu/nu) (6–8 weeks) were obtained from Charles River . The mice were housed in laminar flow cabinets under specific pathogen-free conditions. The University of Nebraska Medical Center Institutional Animal Care and Use Committee approved animal protocols used in this study (IACUC #97-069-03), which comply with the Public Health Service Policy on the Humane Care and Use of Laboratory Animals.−1 . The SW1990 cell line was established from spleen metastasis of grade II pancreatic adenocarcinoma derived from the exocrine pancreas (−1 (Life Technologies Inc.) at 37°C without CO2. For in vivo injections, cells were harvested from subconfluent cultures by treatment with 0.05% trypsin and 0.53 mM EDTA  and resuspended in Hank's balanced salt solution (HBSS) for injection. Only single-cell suspensions with >90% viability were used for injection.The CD18/HPAF cell line used in the study was originally derived from the parental heterogeneous HPAF pancreatic tumour cell line by a limiting dilution technique (−1 medium) at 37°C for 15 min. The digestion of the tissue was terminated by adding 10% DMEM. After washing the tissue three times in DMEM medium, tissue fragments were seeded into six-well plates and incubated at 37°C in a humidified atmosphere of 5% CO2 in air. After 24 h, tumour cells began to migrate out from the tissue pieces into the surrounding areas. The wells became subconfluent at day 5 and were trypsinised with Trypsin-EDTA solution twice for different time periods: first for 1 min to detach and remove the fibroblasts and second for 5 min to harvest the tumour cells. Cells were washed and seeded in flasks containing 10% DMEM medium.A portion of tumour tissue, obtained 2 weeks after implantation of the CD18/HPAF cells into the pancreas or the SC tissue of the nude mice, was placed in a 10% DMEM medium and minced finely with a scalpel. The medium containing the tissue pieces was centrifuged and the supernatant containing the floating fat tissue was removed. The tissue pellet was treated with DMEM supplemented with collagenase P (3.75 mg ml6 cells ml−1. Mice were anaesthetised with 100–200 mg kg−1 ketamine and 5–16 mg kg−1 of xylazine. A volume of 50 μl of cell suspension (10 × 106 cells ml−1) was injected into different tissues like the pancreas, submandibular gland (SMG), ovary, stomach, and SC site. After the injection, the organ was returned to the correct position and the abdomen was sutured using chromic catgut. The skin was closed with metal clips, which were removed 10 days later. The SC injection was performed using 50 μl of cell suspension at a site on the back between the scapulae. After implantation, mice were inspected twice a week. Tumour formation was checked twice a week in the first 2 weeks and daily thereafter. Tumour-bearing mice were killed when an intra-abdominal mass measuring ∼2 cm in diameter was palpated. To assess the tumour dissemination pattern, in each group at least four mice were kept alive until they were moribund. After sacrifice, primary tumours and the metastatic tumours were weighed, measured, and cut into small fragments. These fragments were processed for immunohistochemistry (IHC) or RNA isolation, or were kept in tissue culture medium and processed to obtain a cell line, as described in the previous section.Tumour cells were harvested from the culture flasks by trypsinisation in EDTA solution, and were washed by centrifugation in a serum-containing medium. After being washed twice in PBS pH 7.4 (Life Technologies Inc.), they were resuspended in the same buffer at a concentration of 10 × 10The total cellular RNA from the normal human pancreas, human pancreatic xenografts, and CD18/HPAF cell line was isolated by the guanidine isothiocyanate and cesium chloride (CsCl) cushion ultracentrifugation method (μm) were assayed for MUC4 apomucin by using a modification of the previously described ABC immunohistochemical method  or pre-immune rabbit serum as a negative control. The specificity of the antisera generated against the tandem-repeat peptide in staining pancreatic tumour cells was evaluated as described earlier  from the tumour tissue or cell lines was reverse transcribed using the first-strand cDNA synthesis kit  and oligo d(T) primers, according to the manufacturer's instructions. Oligonucleotide primers to the nontandem repeat region of MUCs 1, 2, 3, 4, 5AC, 5B, 6, 7, TGFβ1, and TGFβ2 are designed from the published sequences in the GenBank, as described earlier . PCR amplification reactions were described previously  or ectopically (SC) in nude mice. After 7 days, at any given time point, the extent of tumour growth was higher at OT compared to SC sites. At 20 days after injection, the OT tumour volume was found to be 2.5-fold higher as compared to the SC tumours, reflected in the tumour weight, as shown in P<0.01) was observed for the OT tumour 6182+1003 mm3 compared to SC tumour 1774+844 mm3. The OT tumours metastasised to LNs in all animals compared to no metastasis in the SC tumours.Similar results were obtained using another pancreatic tumour cell line, SW1990, where a significant difference in tumour volume , tumour tissues, and normal human pancreas was fractionated on agarose gel electrophoresis, Northern blotted, and probed with a MUC4 tandem repeat cDNA probe. As reported in our previous study (MUC4 cDNA probe hybridised to a large-sized transcript (∼26.5 kb) in CD18/HPAF cells, and showed a smear ranging from 10 to 29 kb in the OT tumours on Northern blot , we isolated and cultured cells from the SC tumours, and studied the MUC4 expression. In SC tumour cells cultured MUC4 and TGFβ2 transcripts  without expression of MUC4 (data not shown). On the other hand, the expression of TGFβ1 was found to be similar in tumours (OT and SC) and the CD18/HPAF cells.Previously, we demonstrated a positive correlation in the expression of β2 was investigated. The SW1990 OT exhibited a high level of MUC4 and TGFβ2, as compared to the SW1990 parental line or the SW1990 SC (data not shown).As a control, another cell line, SW1990, was used to validate the results. The SW1990 line was implanted in OT and SC sites in nude mice. Expression of MUC4 as well as TGFMUC4 expression along with other mucin genes, RT–PCR amplification was performed using mucin gene-specific and GAPDH primers designed from the published sequences in the GenBank. A comparison of mucin gene expression in the normal human pancreas tissue, pancreatic tumour cell line (CD18/HPAF), and tumour tissues (OT and SC) is shown in MUC4 in the normal human pancreas and the SC tumours, whereas a high level of MUC4 expression was found in the CD18/HPAF cell line and the OT tumours. The expression of MUC1 and MUC5B appeared similar in all the samples. MUC2 was detected only in the tumour cell line, but not in the normal human pancreas and tumour samples. The expression of MUC5AC was weak in OT and SC tumours, with traces in the cell line and normal human pancreas. MUC6 was detected at a high level only in the normal pancreas. MUC3 and MUC7 mRNA expression was not detected in the CD18/HPAF cell line and tumour samples. The positive controls  for MUC2, MUC3, MUC5AC, MUC5B, and MUC7 showed mucin expression. Among eight mucin genes analysed, MUC4 was the only gene that showed high levels of expression in OT tumours, with no detectable expression in the normal pancreas or SC tumours. For PCR analysis, primers were designed in the non-tandem repeat regions of the human mucin genes. The amplified PCR products for each mucin gene showed the expected size with 100% sequence identity to the corresponding human sequences, thereby ruling out the possibility of amplification of the mouse Muc4.To obtain a comparative picture of neu  rates after implantation at OT sites compared to SC sites in nude mice. In addition, the OT tumours also showed a high incidence of metastasis to regional LNs, and distant metastasis to mediastinal LNs and mesenteric LNs. A high level of MUC4 transcripts and proteins was observed in the human pancreatic tumour xenografts at an OT site, a site analogous to human pancreas that does not show MUC4 expression, compared to SC sites. When tumours were generated at other MUC4-expressing sites in nude mice like SMG and stomach, these tumours also showed equivalent levels of MUC4 (unpublished result). Pancreas, SMG, and stomach, being physiologically active organs that are well perfused compared to the SC environment, have spare vasculature. The MUC4-expressing cells, when grown in the well-vascularised site (OT), revealed a high level of MUC4 expression, as compared to a less-vascularised (SC) environment. The observation suggests a role of serum factors in regulating the MUC4 expression, or there may be a clonal expansion of a non-MUC4-expressing cell type in the SC tumours. To answer these questions, we harvested SC tumour cells and cultured them in vitro. The in vitro culture of SC tumour cells returned the expression of MUC4 transcripts to the parental cell line level, further suggesting a role of serum factor(s) in regulating MUC4 expression. Our earlier study also demonstrates a serum-dependent increase in MUC4 expression in human pancreatic tumour cells  showed an increased expression in many advanced human cancers  in its regulation. Our results also indicated a direct correlation between the MUC4 expression and the levels of TGFβ2 transcripts in the CD18/HPAF tumours, as well as in CD18/HPAF cells in vitro, as described earlier (in vivo conditions.In summary, we believe that this is the first study showing


Counter-irritation (CI) triggered by different non-specific irritant stimuli delayed the growth of a murine tumour of non-detected immunogenicity. The syngeneic LB tumour transplant by itself also induced CI and decreased the number of leukocytes migrating to a secondary s.c. irritant stimulus, e.g. sponge or carrageenan. On the other hand, partial inhibition of cell migration by treatment with either 0.5 mg kg-1 indomethacin or 0.3 mg kg-1 piroxicam retarded LB tumour growth, presumably by a mechanism unrelated to inhibition of immune responses by PGE2. It is suggested that CI may play a role in the early stages of concomitant resistance.


Therapy-induced accumulation of perforin-containing cells at the tumour site was significantly impaired in CD8 KO mice, and marginally in CD4 KO mice. Moreover, CD4 KO mice failed to produce substantial amounts of the tumour suppressive cytokine IFN-γ. This is in sharp contrast to normal mice where a massive local release was recorded. CD8 KO mice displayed a spontaneous production of interleukin (IL)-4 and IL-10 locally in the tumour. Neither normal nor CD4 KO mice produced detectable levels of these Th-2-associated cytokines. The high level of IL-10 was demonstrated to inhibit Fab–SEA tumour therapy, since the therapeutic efficacy was significantly higher in IL-10 KO mice. These results illustrate the importance of a finely tuned cellular collaboration to regulate the various phases of an efficient anti-tumour immune response. © 1999 Cancer Research CampaignTo target T-cells to the tumour area we created a recombinant protein of the bacterial superantigen (SAg) Staphylococcal enterotoxin A (SEA) and the Fab-fragment of a tumour-reactive antibody. This antibody-targeted SAg immunotherapy therapy has been shown to be highly efficient, eliminating > 95% of the pulmonary metastasis in mice carrying established melanoma micrometastases. Earlier studies demonstrated that elimination of the C215-expressing B16-melanoma lung metastasis was dependent on interferon (IFN)-γ release and expression of perforin. In the present study, therapeutic effector functions were analysed both locally at the tumour site and systemically in the spleen. In order to elucidate the role of each T-cell subset during Fab–SEA therapy, CD4 knock-out (KO) and CD8 KO mice were used. Tumour size reduction was statistically significant in Fab–SEA-based tumour therapy in both types of T-cell-deficient mice compared to wild-type mice. CD4 KO mice displayed a drastic reduction in the number of tumour-infiltrating macrophages and CD8


The concentration of HB-EGF in OVCA significantly increased compared to that in OVC or NO (P<0.01). No significant difference in the concentration of TGF-α and amphiregulin was found between the OVCA and NO or OVC groups. In peritoneal fluid, HB-EGF is sufficiently elevated to activate cancer cells even at an early stage of OVCA. These results suggested that HB-EGF in peritoneal fluid might play a key role in cell survival and in the proliferation of OVCA.Epidermal growth factor receptor (EGFR) has been implicated in tumour growth and extension of ovarian cancer. Peritoneal fluid in ovarian cancer patients contains various growth factors that can promote tumour growth and extension. In order to investigate the clinical significance of EGFR ligands as activating factors of ovarian cancer, we examined the cell proliferation-promoting activity and the level of EGFR ligands in peritoneal fluid obtained from 99 patients. Proliferation-promoting activity in peritoneal fluid from 63 ovarian cancer patients (OVCA) was much higher than peritoneal fluid from 18 ovarian cyst patients (OVC) and 18 normal ovary patients (NO), and the activity was suppressed only by antibodies against EGFR or heparin-binding epidermal growth factor (HB-EGF). A large difference was observed in the level of EGFR ligands between HB-EGF and TGF- Ovarian cancer (OVCA) is the most frequent cause of cancer death among all gynaecologic cancers, and in the last 30 years current therapies have not improved cure rates . heparin-binding-EGF like growth factor (HB-EGF), amphiregulin (AR), betacellulin, epiregulin, and epigen  system has been implicated in the pathogenesis of different types of carcinomas , and stored at −80°C until use.All of the 99 patients in this study underwent surgery between 1994 and August 2003 at the Department of Obstetrics and Gynecology, Kyushu University Hospital. Peritoneal fluids were obtained from 99 women, who gave signed informed consent . In all,3H]thymidine (6.7 Ci mmol−1) was obtained from New England Nuclear . Mouse anti-human EGF receptor neutralising antibody and goat anti-human HB-EGF neutralising antibody was obtained from Upstate Inc.  and R&D Systems Inc. , respectively. Goat anti-human TGF-α neutralising antibody, mouse anti-human AR neutralising antibody, mouse anti-human EGF neutralising antibody, goat anti-human epiregulin neutralising antibody and goat anti-human betacellulin neutralising antibody were also purchased from GT . The manufacturer's instructions detail that at least 5 ng ml−1 of EGFR and EGFR ligands  can be neutralized in the use of 10 μg ml−1 of these antibodies, respectively. Polyclonal rabbit anti-EGFR and anti-ErbB-4 antibodies were obtained from Santa Cruz Biotechnology  and Upstate Biotechnology Inc. , respectively. Peroxidase-conjugated goat anti-rabbit IgG was purchased from Zymed .Recombinant human HB-EGF and synthetic LPA were purchased from R&D Systems Inc.  and from Avanti Polar Lipids, Inc. , respectively. thymidine incorporation was examined in SKOV 3 cells, using 10% human peritoneal fluid. The cells were treated in the same manner as the WST-1 assay, and the medium was then replaced with RPMI-1640 plus 10% of each human peritoneal fluid for an additional 24 h in the absence or presence of inhibitory antibodies against EGFR, HB-EGF, TGF-α, amphiregulin, epiregulin, betacellulin, and EGF, or with RPMI-1640 plus 10% peritoneal fluid from patients with a normal ovary (NO) in the absence or presence of various concentrations of LPA or HB-EGF. Peritoneal fluids from 30 patients with OVCA  were available for the experiment using inhibitory antibodies. [3H]thymidine (1 μCi well−1) was then added to the cell culture. After 4 h of labelling with [3H]thymidine, cells were washed with PBS, lysed with NaOH and treated with TCA. After adding scintillation fluid, [3H]thymidine uptake was measured in a β-scintillation counter. Each experiment was conducted in triplicate. The mean value was considered as the representative value for each experiment. The WST-1 assay and the [3H]thymidine incorporation were also examined in other OVCA cell lines including RMG-1 and OVMG1 cells in 10 cases with a NO and in 20 cases with OVCA.SKOV3 cells, derived from OVCA, were maintained in RPMI-1640  supplemented with 10% (v v5) of SKOV3, RMG-1, and OVMG1 were treated in the same manner as in the WST-1 assay. Thereafter, this medium was replaced with the RPMI-1640 plus 10% of each human peritoneal fluid for an additional 24 h in the absence or presence of inhibitory antibodies against HB-EGF. Cells were harvested, pooled, and then fixed with 4% paraformaldehyde and 70% ethanol. After further washing in PBS, cells were incubated with TdT reaction reagent for 1 h at 37°C, according to the manufacturer's recommended protocol . TUNEL-positive cells were quantified as apoptotic cells by flow cytometric analysis .The OVCA cell lines (1 × 10125I-diphtheria toxin (DT) to HB-EGF was measured as described previously  in accordance with the manufacturer's instructions. Samples were analysed in triplicate. Levels of TGF-α and AR were calculated from the linear areas of the standard curves obtained using Multiskan MS, version 8.0 , respectively. The mean value was used as the representative value. The lower limits for detection of TGF-α and AR were 5 and 10 pg ml−1, respectively. When the amount was less than the detection limit, the TGF-α or AR value was recorded as 5 or 10 pg ml−1, respectively.Concentrations of TGF-P-value less than 0.05 was considered statistically significant.The statistical significance was assessed using the Mann–Whitney test and a To investigate the expression of EGFR and ErbB-4 in the OVCA cell lines of SKOV3, RMG-1, and OVMG1, each protein expression was examined using immunoblotting analysis. The expression of EGFR in RMG-1 cells increased remarkably compared to those in SKOV3 or OVMG1 cells. There was little difference in the expression of ErbB4 among these three lines of OVCA cells .3H]thymidine incorporation into DNA. In the absence of the patient's peritoneal fluid, SKOV3 cells showed [3H]thymidine incorporation at a minimum value (5800±1200 cpm). Addition of peritoneal fluid resulted in increased DNA synthesis in all cases, but peritoneal fluid from OVCA patients enhanced DNA synthesis much highly than that from NO and OVC (each P<0.01) (P<0.01). In [3H]thymidine incorporation, the values in the 10 cases with a NO and the 20 cases with OVCA were 5823±1066 c.p.m. (count per minute) and 9241±1553 c.p.m (mean±standard deviation) (RMG-1 cells), and 6903±1134 c.p.m. and 12572±1951 c.p.m. (OVMG1 cells), respectively. In RMG-1 and OVMG1 cells, the DNA polymerisation property mediated by peritoneal fluid in OVCA was also significantly elevated, compared with that in NO (both P<0.01). These results indicate that peritoneal fluid in patients with OVCA possesses a significant cell proliferative property.To assess the cell proliferative property mediated by factors in peritoneal fluid, cell proliferation of SKOV3 cells was examined using the WST-1 assay after incubation with peritoneal fluid. WST-1 assay demonstrated that the value in patients with OVCA significantly increased compared to that in patients with a NO or ovarian cyst (OVC) . To addr P<0.01) . In the 3H]thymidine incorporation was significantly reduced in the presence of anti-EGFR or anti-HB-EGF neutralising antibody, while neutralising antibodies against TGF-α, AR, epiregulin, betacellulin, and EGF had no effect on [3H]thymidine incorporation in SKOV3 cells (3H]thymidine incorporation were 7301±1093 and 7308±1120 c.p.m (RMG-1 cells), and 9513±1510 and 9160±1296 c.p.m. (OVMG1 cells), respectively. [3H]thymidine incorporation was also significantly reduced in the presence of anti-EGFR or anti-HB-EGF neutralising antibodies using RMG-1 or OVMG1 cells.To examine which EGFR ligands contribute to SKOV3 cell proliferation in a patient's peritoneal fluid, the effects of peritoneal fluid on SKOV3 cell DNA synthesis were measured in the absence or presence of an inhibitory antibody against EGFR or each EGFR ligand. [V3 cells . The conP<0.01)  were detected as TUNEL-positive under serum-free conditions . In a paP<0.01) . These rμM (μM) did not show any growth-promoting effect in the present cell system  (α levels were quite low (less than 40 pg ml−1) in all cases, and 46 out of 99 cases  showed levels lower than the detection limit (5 pg ml−1). Amphiregulin levels were scattered among the cases, but were less than 1000 pg ml−1 except in three OVCA cases. No significant differences were found in TGF-α and AR levels among the patients with NO, OVC, and OVCA  . Transfoand OVCA . In addiα and AR , suggestα and AR were observed between pre- and post-chemotherapy  Changes in the amount of HB-EGF were reflected by therapeutic efficacy in OVCA patients. Taken together, the present study suggests that HB-EGF plays a pivotal role in tumour growth and extension of OVCA. In this study, however, cell proliferative activities in SKOV3, RMG-1, or OVMG1 cells, which were mediated by peritoneal fluid from OVCA patients, were not always dependent on the expression of EGFR or ErbB-4. It remains open to debate how EGFR or ErbB-4 is involved in the cell proliferation mediated by peritoneal fluid in OVCA patients.In this study, we have shown the following: (1) HB-EGF levels in peritoneal fluid were elevated at all stages and at recurrence, and levels of HB-EGF were sufficient to proliferate and to allow survival of OVCA cells, whereas the levels of the other five EGFR ligands in peritoneal fluid had little effect on the proliferation of OVCA cells. (2) Cell proliferation of OVCA cells was stimulated by the addition of HB-EGF, but not LPA, in Among the members of EGF family growth factors, HB-EGF, AR, and betacellulin have heparin-binding properties . IncreasIn OVCA, LPA regulates the production of LPA itself through the activation of phospholipase D and phospholipase A2 . ConsideHeparin-binding epidermal growth factor in peritoneal fluid is generated mainly from cancer cells and peritoneal mesothelial cells, which have the same origin as coelomic epithelium of the NO. Our study showed that HB-EGF levels were already elevated even in the ascitic fluid of patients with stage Ia OVCA, whereas OVCA cells are enclosed within the cyst wall. This suggests that HB-EGF is not secreted from cancer tissues in an early stage. In peritoneal mesothelial cells, HB-EGF is constitutively expressed and produced by cytokine stimulation (These results are the first demonstration of HB-EGF as an OCAF. Heparin-binding epidermal growth factor is known to enhance cell motility as well as cell proliferation. Therefore, elevated HB-EGF in peritoneal fluid may contribute to not only survival and proliferation of cancer cells, but also to their dissemination into the peritoneal cavity. It is easier to measure HB-EGF levels in peritoneal fluid compared with LPA, suggesting that HB-EGF is a promising bioactive marker of OVCA. For therapy, relevant LPA antagonists have yet to be developed, while Iressa, a specific EGFR tyrosine-kinase inhibitor, is not effective in OVCA (


Tumour angiogenesis, described by Folkman in the early seventies, is an essential, complex, and dynamic process necessary for the growth of all solid tumours. Among the angiogenic factors secreted by the tumour cells, the Vascular Endothelial Growth Factor (VEGF) is one of the most important. Most types of human cancer cells express elevated levels of this proangiogenic factor and its receptors. New molecules, called anti-angiogenic, are developed to impair VEGF pathway and tumour vasculature. Despite important results, the clinical benefits of anti-VEGF therapy are relatively modest and usually measured in weeks or months. Why following anti-angiogenic therapy do some patients respond transiently and then why does tumour grow again and disease progress and which compensatory mechanisms could explain the anti-angiogenic treatment failure? Tumour angiogenesis, as described by Folkman in the early seventies and confirmed today , is an eFor example, Bevacizumab , a humanized neutralizing monoclonal antibody against VEGF, is the first anti-angiogenic molecule approved by the FDA in 2004. This antibody, which is becoming an important anti-angiogenic treatment, is commonly used in association with a cytotoxic chemotherapy for late stages of colon cancer, nonsmall-cell lung cancer, breast cancer, glioblastoma, and metastatic renal cell carcinoma (mRCC). Sorafenib tosylate  and Sunitinib malate , two small tyrosine kinase inhibitors (TKI), target multiple signaling pathways including VEGF and platelet-derived growth factors (PDGFs). Sorafenib and Sunitinib are approved by the FDA to treat mRCC as a single agent. Sunitinib is used in gastrointestinal stromal tumours (GISTs) and Sorafenib is used for treating patients with liver cancer. Every year a variety of new anti-angiogenic molecules are developed with more than 800 clinical trials .Despite important results, the clinical benefits of anti-VEGF therapy are relatively modest and usually measured in weeks or months . In someThe fraction of nonresponsive patients included in anti-angiogenic clinical trials such as anti-VEGF antibody treatment or tyrosine kinase inhibitors is significant , 15, 21.α (TNF-α) [The angiogenic redundancy is the first resistance mechanism identified following anti-VEGF therapy , 23. VEG (TNF-α) –26  treatment, the vascular density of tumours is reduced and tumours have regions of acute hypoxia. Histological analysis shows that tumours have a wide front of tumour invasion compared to controls and tumours are more invasive after one week of treatment and this increases after four weeks of continuous treatment. Despite the disruption of VEGF angiogenic switch following long-term treatment, a phenotypic resistance to anti-VEGFR2 therapy emerges. During this phase, the tumour's revascularization is increased indicating an active tumour angiogenesis. This progression phase is linked to enhanced production of redundant angiogenic factors, such as FGF family members. If mice are treated with anti-VEGFR, then with FGF-trap just before tumour vasculature regrowth, the tumour growth and neovascularization are attenuated. This independence of VEGF pathway is associated with hypoxia-mediated induction of proangiogenic factors such as FGF-1 and 2, ephrin A1 and 2, and angiopoietin 1 . A synerAntiangiogenic therapies reduce and normalize tumour vasculature but increase intratumour hypoxia , 32 Fig. Hypoxiaα), two genes controlled by HIF-1, and viable circulating endothelial cells [The treatment of recurrent glioblastoma patients with a tyrosine kinase inhibitor targeting VEGF receptors initially leads to disease stable but resistances appear after few weeks. The development of resistance following VEGF blockade is associated with an increase of circulating levels of basic FGF, stromal cell-derived factor 1 alpha  is a potent mitogenic, motogenic, morphogenic factor, and also an important actor in angiogenesis and tumour growth –39. HGF The plasma-membrane bound Notch receptor ligand, Delta-like ligand 4 (DLL4), can be an alternative angiogenic pathway which participates to anti-angiogenic treatment failure. DLL4 is highly expressed by vascular endothelial cells and induced by VEGF. It interacts with Notch cell-surface receptors to act as a negative feedback inhibitor downstream of VEGF signaling to restrain the sprouting and branching of new blood vessels , 48. InhThe angiogenic inhibitors normalize tumour vasculature, reduce tumour size, but increase local hypoxia . It has α, and granulocyte colony-stimulating factor (G-CSF), three factors implicated in CD11b+Gr1+ cells mobilization and activation [Antiangiogenic therapies normalize vessels but increase intratumoral hypoxia which lead to recruitment of bone marrow derived cells  . These ctivation , 59. G-Ctivation . Moreovetivation , 61. Stutivation –64. α expression by endothelial cells is increased by HIF-1 following hypoxic conditions. BMDCs expressed the SDF-1α receptor CXCR4 and are recruited to the ischemic tissue by cell tropism to SDF-1α. SDF-1α upregulation is associated with anti-angiogenic treatment resistance in patients with glioblastoma [α and G-CSF are increased dose-dependently in healthy mice and cancer patients [α and IL-6, in patients treated with Sunitinib, is associated with a poor outcome [α are increased in patients with rectal cancer and seem to be associated with distant metastasis after three years [α be considered as a biomarker of response or resistance to anti-angiogenic treatment [α and Lysyl Oxidase gradient, CD11b+Gr1+ cells will be recruited at premetastatic sites and promote tumour metastasis through matrix metalloproteinase 2 (MMP2) production [Furthermore, SDF-1blastoma . Followipatients . Thereby outcome . During ee years . Could Sreatment ? In respoduction , 69. In oduction . CD11b+Goduction  and matroduction .β [Tumour-associated endothelial cells and pericytes were generally assumed to be genetically stable and to have a low mutational rate . Antiangβ . Endotheβ , 76. Perβ . These cβ . Pericytβ , 79, 80.β . Anti-VEβ . Furtherβ , 83–85. β  (Figure β . VEGFR-2β . Despiteβ , 88. In β . Targetiβ . Moreoveβ . ContrarPatients treated by anti-angiogenic therapy are mainly patients with metastases for which other treatments are no longer available. In vasculature-rich organs such as brain, liver, and lung, primary tumours cells and metastases could coopt with the neighboring quiescent normal blood vessels  Figure . In rat The establishment of a new vascular network by angiogenic process is one part of the basis of solid tumour development and dissemination of tumour cells in the organism as metastasis. The core of the tumour rapidly undergoes hypoxic and tumour cells counteract this process by producing angiogenic factors responsible for growth and migration of quiescent endothelial cells of proximal blood vessels. Among the angiogenic factors, the VEGF is one of the most important. Targeting the VEGF/VEGFR pathway represents a major advance in cancer treatments and an important therapeutic option. Despite the transient effect of these expansive anti-VEGF treatments, the enduring clinical responses are rare. Pre-clinical and clinical trials suggest a host response to VEGF inhibition implicated in treatment failure and can participate to progression of secondary disease. Furthermore, the mechanisms of action of anti-angiogenic drugs and resistances against these molecules vary according to the tumour. Resistance or evasion to anti-angiogenic therapies in preclinical models is more and more reported. For example, anti-angiogenic treatments not only normalize tumour vasculature but also reduce pericyte coverage and increase tumour hypoxia and tumour cells can acquire a more invasive; however the molecular mechanisms are not fully understood. Hence, it is crucial to highlight molecular mechanisms or actors implicated in this phenomenon of resistance in order to anticipate the best responders to the treatment and to improve anti-angiogenic drugs or to develop new agents. Tumours produce multiple factors and animal models suggest that different pathways are activated under or following anti-angiogenic therapies. Hence, targeting complementary pathways implicated in tumour angiogenesis would be more efficient. At last, it is also important to find predictive biological markers of objective response or involved in resistance to anti-angiogenic drugs in order to improve therapy efficacy or to propose alternative anti-angiogenic therapy in case of treatment failure.


A study has been made of the natural history of metastasis of a spontaneous murine squamous carcinoma implanted into syngeneic recipients--a situation where biologically different tumours and variable "host resistance" are not complicating issues. The time distrubution of deaths from metastatic disease was incompatible with a log-normal distribution but was accurately described by an exponential pattern of survival following an initial lag. While the average life of doomed mice correlated with predictions based on growth rates, there was a wide range of survival times indicating random influences on the evolution of metastitic disease. Insofar as tumours which grew to 20 mm3 or less in 5 days after tumour cell injection failed to initiate metastases, while tumours which reached a size of 120 mm3 or greater (irrespective of duration) produced metastases in 38/39 mice, tumour size was a prognostic index. However, within the size range 33-150 mm3 the correlation between metastatic risk and size was not statistically significant. No correlation between metastatic risk and duration of tumour growth from 6 to 29 days was observed. Two integral functions of tumour size and duration were tested but neither gave a better correlation with metastatic risk than did size alone.


The efficacy of radioimmunotherapy of tumours with radiolabelled monoclonal antibodies (MAbs) depends on the amount of antibody taken up by the tumour and on its intratumoral distribution. In the case of MAbs directed against intracellular antigens, increasing the permeability of the cytoplasmic membrane may augment the bioavailability of the antigen for the antibody. This raises the question whether the induction of tumour necrosis by chemotherapy can enhance the tumour uptake of radiolabelled monoclonal antibodies. In this work, the effect of doxorubicin on the biodistribution of Po66, an MAb directed against an intracellular antigen, was studied in nude mice grafted with the human non-small-cell lung carcinoma cell line SK-MES-1. After injection on day 0 of 125I-labelled Po66, tumour radioactivity increased up to days 3-5, and then remained unchanged to day 14. The combined administration of 125I-labelled Po66 with 8 mg kg-1 doxorubicin, in two doses separated by 7 days, doubled the radioactivity retained by the tumour. Histological and historadiographic analysis showed, however, that the drug induced cellular damage. In the absence of doxorubicin, the accumulation of Po66 was restricted to some necrotic areas, whereas with doxorubicin the necrosis was more extensive and the antibody more evenly distributed. These results suggest that chemotherapy and immunoradiotherapy combined would enhance tumour uptake of radioisotope and promote more homogenous distribution of the radiolabelled MAb. This would promote eradication of the remaining drug-resistant cells in tumours.