Phagocytosis of apoptotic cells by macrophages induces a suppressor phenotype. Previous data from our group suggested that this occurs via Platelet-activating factor receptor (PAF-R)-mediated pathways. In the present study, we investigated the impact of apoptotic cell inoculation or induction by a chemotherapeutic agent  on tumour growth, microenvironmental parameters and survival, and the effect of treatment with a PAF-R antagonist (WEB2170). These studies were performed in murine tumours: Ehrlich Ascitis Tumour (EAT) and B16F10 melanoma.Tumour growth was assessed by direct counting of EAT cells in the ascitis or by measuring the volume of the solid tumour. Parameters of the tumour microenvironment, such as the frequency of cells expressing cyclo-oxygenase-2 (COX-2), caspase-3 and galectin-3, and microvascular density, were determined by immunohistochemistry. Levels of vascular endothelium growth factor (VEGF) and prostaglandin E2 (PGE2) were determined by ELISA, and levels of nitric oxide (NO) by Griess reaction. PAF-R expression was analysed by immunohistochemistry and flow cytometry.in vivo pre-treatment with WEB2170. This treatment also reduced tumour growth and modified the microenvironment by reducing PGE2, VEGF and NO production. In B16F10 melanoma, WEB2170 alone or in association with DTIC significantly reduced tumour volume. Survival of the tumour-bearing mice was not affected by WEB2170 treatment but was significantly improved by the combination of DTIC with WEB2170. Tumour microenvironment elements were among the targets of the combination therapy since the relative frequency of COX-2 and galectin-3 positive cells and the microvascular density within the tumour mass were significantly reduced by treatment with WEB2170 or DTIC alone or in combination. Antibodies to PAF-R stained the cells from inside the tumour, but not the tumour cells grown in vitro. At the tissue level, a few cells (probably macrophages) stained positively with antibodies to PAF-R.Inoculation of apoptotic cells before EAT implantation stimulated tumour growth. This effect was reversed by We suggest that PAF-R-dependent pathways are activated during experimental tumour growth, modifying the microenvironment and the phenotype of the tumour macrophages in such a way as to favour tumour growth. Combination therapy with a PAF-R antagonist and a chemotherapeutic drug may represent a new and promising strategy for the treatment of some tumours. It has been suggested that M1 and M2 represent the extremes of a variety of phenotypes that macrophages can express and that the M2 phenotype is associated with suppressor functions  reportedcascades .in vitro and reduced the formation of new vessels in tumours induced by implantation of these cells [The findings that apoptotic cells share common ligands with PAF, that apoptotic cells dampen macrophage activation and that PAF-R is somehow involved in these effects could be particularly relevant in the case of tumour growth. As the number of tumour cells increases during tumour development, many of these cells die by apoptosis or necrosis due to the reduction in oxygen and nutrient supply. Induction of apoptosis is also the mechanism of action of anti-tumour chemotherapy. Correa et al  clearly se cells ; in B16Fse cells ; in EAT se cells .Based on the data discussed above we postulated that the interaction of macrophages with apoptotic cells in the tumour microenvironment, through PAF-R-dependent mechanisms, would drive macrophage polarization towards a suppressive phenotype favoring tumour growth. In the present study we investigated this hypothesis in two murine tumours: EAT and melanoma B16F10. Apoptotic cells were either inoculated into the tumour or induced by dacarbazine (DTIC), an agent that is widely used in human melanoma therapy. The role of the PAF-R was evaluated using the antagonist WEB2170. Tumour growth was evaluated by counting the cells in the ascitis or measuring tumour volume. The tumour microenvironment elements that were analysed were the levels of PGE2, VEGF and NO in the ascitis and, in the melanoma, the number of cells expressing COX-2, the number of intra-tumoural blood vessels and tumour infiltration by activated macrophages/dendritic cells expressing galectin-3, which is associated with the suppressive M2 phenotype. PAF-R expression was evaluated in cells from inside the tumour and in melanoma cells taken from cultures.Seven- to 10-week-old male BALB/C and 7- to 8-week-old female C57BL/6 mice from our own animal facilities were used. All animal procedures were in accordance with the ethical principles adopted by the Brazilian College of Animal Experimentation and approved by the Ethical Committee for Animal Research of the Institute of Biomedical Sciences, University of São Paulo.2. Ehrlich ascitis tumour (EAT) cells were grown as described elsewhere [The murine melanoma cell line B16F10 was a kind gift from I. Fidler  and was cultured in RPMI 1640 medium , pH 7.4 supplemented with 10% fetal bovine serum , in the absence of antibiotics, at 37°C and 5% COlsewhere .3-105 cells) was injected intraperitoneally (i.p.) in BALB/c mice. At different times after tumour implantation, aliquots of the ascitis fluid were recovered using a 21-gauge needle to measure cell number and mediator/cytokine levels. B16F10 melanoma cells (5 × 105) were injected subcutaneously (s.c.) in C57BL/6 mice and tumour growth was determined via measurement of the diameter of the solid tumour mass, from which the volume was estimated using the formula for a spheroid: V = 0.52 × (largest axis) ×  2.Ehrlich ascitis tumour  was injected i.p. 30 minutes before tumour implantation and injections were repeated every 24 h until the end of the experiment. In order to evaluate the possible benefit of the combined therapy with WEB2170, treatment with the chemotherapeutic drug DTIC was chosen at a suboptimal regimen based on previously published studies ,14. In og for 10 min) and VEGF levels were determined in the supernatant by ELISA using goat polyclonal antiserum against mouse VEGF . PGE2 levels were also determined by ELISA using a commercially available kit (Cayman Chem. Co.). NO levels were determined according to the method described by Bartholomew (1984) [4 solution for 10 minutes at 4°C. Three μL of 2.5 N NaOH were added and the ascitis fluid was incubated at 4°C for 10 minutes. The NO3- content was converted to NO2- through addition of 15 μL of 0.5 M NaH2PO4, 15 μL of 2.4 M ammonium formiate and 10 μL of a suspension of E. coli to the ascitis fluid. The bacterium E. coli expresses nitrate reductase, which catalyses the conversion of nitrate to nitrite. Samples were further incubated at 37°C for 2 h, centrifuged (18000 × g for 5 min at 20°C), and 100 μL of Griess Reagent  were added to the same volume of the supernatant. NO levels were quantified in a spectrophotometer at 540 nm.At day 10 of EAT growth, BALB/C mice were sacrificed and the ascitis fluid was recovered. The suspension was centrifuged (180 × w (1984) . Briefly3 EAT cells and sacrificed after 7 or 10 days. The peritoneal cavity was injected with 3 mL of PBS to recover the ascitis. The fluid was centrifuged (180 × g for 10 min) and the cell pellet resuspended in 1 ml of RPMI 1640 with 10% fetal calf serum. Trypan Blue dye (0.2%) was added to aliquots of cellular suspensions and the percentage of cells that had taken up the dye (necrotic cells) was determined microscopically. The percentage of apoptotic cells was determined by flow cytometry  after incubation of 3 to 5 × 105 cells with propidium iodide (20 μg/mL in 0.1% sodium citrate and 0.1% Triton X-100) at 4°C for 2 h in the dark.BALB/C mice were inoculated i.p. with 1 × 10g, 4°C, 7 min), the pellet was resuspended in RPMI medium and cell viability was determined using Trypan Blue vital dye. After this, thymocytes were γ-irradiated (450 rad) and incubated at 37°C for 18 hours. Apoptotic cells were detected by annexin V staining and the percentage of apoptotic cells was measured by flow cytometry , as described elsewhere [6 viable or apoptotic thymocytes into the peritoneal cavity 2 h before the inoculation of 1 × 105 EAT cells. Animals were sacrificed after 5 days and aliquots of the ascitis fluid were recovered to estimate EAT growth via direct cell counting in a Neubauer chamber under a light microscope.Murine thymocytes were obtained through surgical extraction of the thymus from BALB/C mice. After thymus dissociation, the thymocyte suspension was centrifuged , PAF-R , COX-2  and activated caspase-3 . Reactions were performed as described elsewhere . Images Tumours were excised and kept in RPMI 1640 at 4°C for immediate processing. All collected tumours were washed in ice-cold PBS, finely minced and transferred to 60 mm-diameter Petri dishes containing 1 mL MTH  supplemented with 0.15 Wunsch units/mL of Liberase Blendzyme 3 (Roche). Fragments were incubated for 30 minutes at 37°C under constant agitation (80 rpm) for mechanical dissociation. Cell suspensions were then filtered using nylon membranes (41 μm pores). The filtrate was centrifuged at 4°C (1200 rpm for 5 min), washed in cold PBS and resuspended in 3 mL of red blood cell lysis solution (Amersham Biotech). After 3 min, another 3 mL of RPMI 1640 was added to the suspension, which was then centrifuged as above and washed in PBS. Cell number was then determined by direct counting using a Neubauer chamber.For flow cytometry experiments, a million cells were separated for each reaction tube or well. Cells were washed with PBS-BSA (PBS containing 0.5% BSA) and then incubated with an Fc blocker antibody for 15 min at 4°C. Cells were then centrifuged (1200 rpm for 5 min at 4°C), washed with PBS-BSA and incubated with 0.3 μg/mL of a rabbit polyclonal anti-PAF-R antibody  for 60 min on ice. After washing, cells were incubated with a FITC-labeled anti-rabbit IgG for 30 min on ice in the dark. After washing, the cells were then suspended in 2% formaldehyde in PBS and kept in the dark until acquisition and analysis in a flow cytometer . Histogram analysis was done using the CellQuest software. The control samples were incubated with the Fc blocker and with the FITC-labeled secondary antibody.Data were analysed using ANOVA followed by Tukey or Bonferroni post-hoc tests. Survival was analysed by the Kaplan-Meier method and survival curves were compared using the log-rank test. Differences were considered significant when p < 0.05.3 tumour cells into the peritoneal cavity of BALB/c mice. The number of cells present in the ascitis fluid was counted on days 1, 7 and 10 after implantation. It can be seen that the number of cells in the peritoneal cavity increased progressively. Whereas at day 7, around 92% of the cells were EAT cells, at day 10 virtually all cells identified in the differential counts were EAT cells. Treatment with the PAF-receptor antagonist WEB2170 (5 mg/kg) significantly reduced the number of tumour cells, markedly after 10 days of treatment . The results showed that previous inoculation of apoptotic but not live thymocytes increased EAT growth, as total cell counts were about 2-fold higher (p < 0.05) in this group than in the control group . Mice were treated with WEB2170, 30 min before tumour implantation and daily up to the 6th day. The levels of PGE2  or WEB2170 (76%). The combined treatment with both DTIC and WEB2170 was around three times more effective than either treatment alone.The pro-tumoural effect observed with the addition of apoptotic thymocytes before EAT implantation prompted us to evaluate a more realistic scenario, namely the impact of blocking PAF-R-dependent pathways during the course of chemotherapy. DTIC (40 μg injected i.p. every 3 days after tumour implantation) was chosen as the chemotherapeutic drug, based on its capacity to induce apoptosis in a wide variety of tumour cells in o models . It can 5) were injected s.c. in C57Bl/6 mice. Fig. The effect of treatments with WEB2170 and DTIC was then evaluated in a solid tumour, the B16F10 murine melanoma model. Tumour cells  within the tumours Fig. , the relApoptotic cells were quantified using antibodies reacting with activated caspase-3. The frequency of caspase-3 positive cells within the tumour was slightly but significantly lower in the group receiving both DTIC and WEB2170. No significant changes were found in the other groups Fig. . Althougin vitro , and this was accompanied by a significant reduction in EAT growth [in vivo was used in our experiments. Participation of PAF-R was also observed in a solid tumour where WEB2170 significantly reduced the growth of melanoma B16F10.These data suggest that PAF-R-dependent mechanisms are able to modify tumour microenvironment elements, including tumour macrophages, during EAT growth. In previous studies in our laboratory, we observed that whereas macrophages taken from the normal peritoneal cavities of mice were able to produce hydrogen peroxide and spread across a glass surface, those taken from mice with EAT retained the round morphology characteristic of non-activated macrophages and were unable to produce hydrogen peroxide . HoweverT growth . This waT growth . The samAs tumours grow, a number of tumour and host cells die. The process of cell death is accompanied by oxidation of membrane phospholipids. We have previously shown that apoptotic cells express PAF-like molecules, which share a common or related receptor with oxLDL and PAF in macrophages . We thusAlthough treatment of melanoma-bearing mice with WEB2170 reduced melanoma growth it did not prolong survival, indicating that the efficacy of treatment was limited to a narrow time window in the initial phase of tumour growth. Another possibility that has to be considered is that increased survival could be related to inhibition of tumour metastasis . Indeed,Blocking PAF-R also modified other elements of the tumour microenvironment such as the increased levels of PGE2, NO and VEGF produced during EAT growth. Moreover, in melanoma the PAF-R antagonist reduced the proportion of COX-2-expressing cells, the microvascular density, and the proportion of activated macrophages/dendritic cells expressing galectin-3 within the tumour microenvironment. These alterations did not contribute to the control of the melanoma, unless the animals were given the chemotherapeutic agent DTIC. Interestingly, as shown in Fig. in vitro, which indicates that the receptor is expressed by cells from the tumour microenvironment rather than the tumour cells themselves.We have shown the expression of PAF-R in homogenates of melanoma but not in melanoma B16F10 cells kept Clearance of dead cells by professional phagocytes such as macrophages may lead to the secretion of cytokines that favour tumour growth . The endThe data suggest that PAF-R-dependent pathways are activated during experimental tumour growth, modifying the phenotype of tumour macrophages and the microenvironment in such a way as to favour tumour growth. Combination therapy with a PAF-R antagonist and a chemotherapeutic drug may represent a new and promising strategy for the treatment of some tumours.The authors declare that they have no competing interests.SIO carried out the Ehrlich tumour protocols, participated in the melanoma protocols, performed the statistical analysis, participated in the discussion of the results and drafted the manuscript. LNSA designed and carried out the melanoma protocols and participated in the discussion of the results. ACO evaluated the PAF-R expression in melanoma cells. SN carried out the immunohistochemistry. PDF carried out the NO measurements and discussed the NO and PGE2 assay data. MCP participated in the design of the melanoma protocols and discussion of the results. CBSR carried out the experiments on galectin-3 expression. RC and SJ conceived, designed and coordinated the study, discussed the data and critically revised the manuscript. Finally, all the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/200/prepub


High-mol.-wt levan injected locally inhibits the growth of Lewis lung carcinoma in C57BL mice. The inhibition is dependent on the number of tumour cells injected and on the dose of levan. The inhibition decreases tumour incidence and size as well as prolonging survival. The polysaccharide is most effective when injected daily beginning on the day of tumour-cell inoculation. Treatment begun on later dates is less effective. Treatment begun one day before tumour-cell inoculation enhances tumour growth. Histological studies showed that levan induces an intense polymorphonuclear (PMN) reaction followed by accumulation of vacuolated, levan-laden macrophages. Both PMN and activated macrophages seemed to have an inhibitory effect upon the growth of the tumour. The effector role of PMN was not explained by the histological study. Tumour cells in close contact with levan-laden macrophages appeared mostly necrotic. Administration of levan begun one day before tumour-cell inoculation produced a similar reaction, but the infiltrating cells did not appear to approach and damage the tumour cells.


An animal tumour model that mimics the human counterpart is essential for preclinical evaluation of new treatment modalities. The objective of this study was to develop and characterize such a model. To accomplish this, the established AY-27 rat bladder transitional cell carcinoma (TCC) cell line was transplanted orthotopically into Fischer CDF344 female rats. AY-27 TCC cells were grown in monolayer cell culture and instilled intravesically as single cell suspensions into bladders that had been conditioned with mild acid washing. Tumour growth was assessed weekly by subjecting the rats to magnetic resonance imaging (MRI). At intervals following implantation and MRI tumour detection, the animals were sacrificed for necropsy, histological examination and immunocytochemical studies. Flow cytometry was also performed for detection of Fas or Fas-ligand expression on AY-27 cells. The overall tumour establishment was 95% (97/102 rats) at 12–50 days, while in a subgroup of animals sacrificed at 16 days, 80 out of 82 animals (97%) developed TCC, the majority of which was superficial. Tumour stage was assessed by gross pathology and light microscopy. Histological examination of the tumour specimens confirmed the presence of grade II–III TCC. Immunocytochemistry confirmed that the tumour model maintained the features of TCC. The changes seen on MRI correlated well with the extent of tumour invasion identified histologically. Patchy carcinoma in situ could be detected histologically 12–13 days post-inoculation, and progressed to papillary tumour or invasive disease thereafter. Neither Fas nor Fas-ligand was expressed on AY-27 cells. The orthotopic AY-27 TCC model is highly reproducible and is ideal for preclinical studies on experimental intravesical therapies. © 1999 Cancer Research Campaign


Angiogenesis is crucial for maintaining the supply of oxygen and nutrients required to support solid tumour growth. Inhibitors of tumour blood vessel formation are therefore being sought, in particular, inhibitors of vascular endothelial growth factor-A (VEGF)-signalling, which has a pivotal role in stimulating neovascular growth and survival. ZD6474 is an orally bioavailable inhibitor of VEGF receptor-2 tyrosine kinase activity that in preclinical studies has been shown to inhibit both VEGF-induced signalling in endothelial cells and tumour-induced angiogenesis. Consistent with inhibition of angiogenesis, once-daily oral dosing of ZD6474 produced significant broad-spectrum antitumour activity in a panel of histologically diverse human tumour xenografts. In addition to its antiangiogenic properties, ZD6474 also has activity against the epidermal growth factor receptor (EGFR) tyrosine kinase, which could impart a direct inhibitory effect on tumour cell growth and survival. This may be particularly relevant in tumours with a dependency upon EGFR signalling, for example in certain tumours harbouring activating mutations in EGFR. RET kinase has also been identified as a third target for ZD6474. This review summarises preclinical studies with this unique agent and considers its future direction in cancer treatment. In order to grow beyond a few millimetres in size, tumours must develop a functioning vascular network to provide essential nutrients and oxygen. Early in tumorigenesis, an ‘angiogenic switch’ occurs within the tumour microenviro nment, which is associated with the altered expression of a range of proangiogenic growth factors, such as vascular endothelial growth factor-A (VEGF) and basic fibroblast growth factor (bFGF). The resulting imbalance of proangiogenic factors stimulates the sprouting of new blood vessels from pre-existing vessels within the surrounding normal tissue. These new vessels grow and infiltrate into the tumour, providing nutrients and oxygen to sustain tumour expansion, and a route for cancer cell dissemination. The carefully controlled balance of pro- and antiangiogenic growth factors during physiological angiogenesis  produces well-ordered vascular structures with mature, functional blood vessels. In contrast, pathological angiogenesis associated with tumour growth is characterised by disorganised vasculature that lacks clear hierarchical organisation, with associated abnormalities in blood flow, vascular permeability and pericyte–endothelial cell interactions, and with a high fraction of immature, proliferating endothelial cells.While many factors have been implicated in promoting angiogenesis, VEGF appears to play a pivotal rate-limiting role, which has been attributed to its ability to regulate key steps in the angiogenesis cascade, including inducing endothelial cell proliferation, migration, survival and capillary tube formation . VasculaVascular endothelial growth factor binds to the receptors VEGFR-1 (Flt-1) and VEGFR-2  on vascular endothelial cells. Significantly, activation of VEGFR-2 alone is necessary and sufficient to effect VEGF-induced mitogenesis, angiogenesis and vascular permeability . These pRecognition of the key role of VEGF-mediated VEGFR-2 signalling in pathological angiogenesis has led to the development of various targeted strategies designed to inhibit VEGFR-2 activation, which include ligand sequestration, receptor antagonist approaches or receptor tyrosine kinase inhibition , thus stimulating EGFR expression in tumour-associated endothelial cells and setting up a paracrine endothelial cell survival signalling pathway that is sensitive to inhibitors of EGFR signalling  tyrosine kinase activity. The EGFR autocrine signalling pathway is central to cancer progression, and overexpression of EGFR and/or its ligands, transforming growth factor (TGF)-α and EGF has been reported in many human tumours . The conConsequently, ZD6474 has the potential to inhibit two key pathways in tumour growth by (i) targeting tumour growth indirectly, via inhibition of VEGF-dependent tumour angiogenesis and VEGF-dependent endothelial cell survival, and (ii) targeting tumour growth directly, via inhibition of EGFR-dependent tumour cell proliferation and survival .50=40 nM). Compared with the IC50 for VEGFR-2 kinase, ZD6474 was only 2.7-fold less active vs VEGFR-3 (Flt-4) kinase, but demonstrated 40-fold selectivity vs the kinase associated with VEGFR-1. Vascular endothelial growth factor receptor-3 and its ligands (VEGF-C and VEGF-D) are known to play key roles in the regulation of lymphangiogenesis (The ability of ZD6474 to inhibit tyrosine kinase activity was determined using recombinant enzyme assays (ogenesis . Althoug50=500 nM), but demonstrated excellent selectivity for VEGFR-2 kinase vs structurally related receptor tyrosine kinases, such as c-kit, and kinases from other families  proliferation in vitro (IC50=60 nM) (50=170 nM). In contrast, much higher doses of ZD6474 were required to inhibit bFGF-stimulated HUVEC proliferation (IC50=800 nM) or basal (serum-stimulated) HUVEC proliferation (IC50>3000 nM). These data demonstrate that ZD6474 is a selective inhibitor of VEGF- and EGF-stimulated cell proliferation.Consistent with its activity in vitro evidence shows that ZD6474 can also elicit direct inhibition of tumour cell growth  was shown to be hypersensitive to ZD6474 (IC50=90 nM for serum-stimulated tumour cell growth). PC-9 has a 15 bp deletion mutation in the gene encoding the EGFR. The EGFR tyrosine kinase of this mutated gene is active even in the absence of exogenously added ligand, and is more sensitive to the inhibitory effects of ZD6474 than wild-type EGFR tyrosine kinase  and selective inhibition of RET-dependent thyroid tumour cell growth in vitro (RET gene (mutation or translocation) that lead to RET receptor signalling-dependent tumour cell growth  cells  is a technique that can be utilised to measure tumour haemodynamic parameters −1 produced significant, dose-dependent inhibition of tumour growth  antitumour effect rather than direct antiproliferative effects on the tumour cell. To support this, we have shown that tumour models that are known to be either sensitive  or insensitive  to the selective EGFR tyrosine kinase inhibitor gefitinib, are all sensitive to ZD6474 , chronic once-daily oral administration of ZD6474 at doses of 25−100 mg kgo ZD6474 .in vitro data indicated that VEGF expression was upregulated in EGFR inhibitor-resistant cells. Gefitinib-resistant tumour cells were crossresistant to ZD6474 in vitro, but not in vivo. This suggests that in these tumour cells the in vitro antitumour activity of ZD6474 is dependent on EGFR tyrosine kinase inhibition, whereas the in vivo antitumour activity of ZD6474 is not. Collectively, these data suggest that ZD6474 may be an effective treatment against tumours with acquired or intrinsic EGFR resistance, because of its ability to inhibit VEGF signalling. Nonetheless, additional inhibition of EGFR tyrosine kinase may afford further therapeutic benefits, depending upon the tumour type. To date, this has been most profoundly shown in a preclinical in vivo study examining established PC-9 human lung cancer xenografts. ZD6474 (12.5–50 mg kg−1 day−1) caused robust regression of PC-9 tumours at all doses . Organ–tumour interactions at the site of the primary tumour and at the site(s) of metastasis are considered important determinants of tumour growth and development in man. Therefore, it has been suggested that orthotopically implanted tumours may recapitulate the natural tumour setting more accurately than subcutaneous models . In orthMin) mice, which develop spontaneous early intestinal adenomas (−1 day−1 for 4 weeks) produced a 75% reduction in overall tumour burden compared with vehicle treated controls , suggesting that ZD6474 could be an effective treatment for early-stage disease. Indeed, early administration of ZD6474 has also been shown to significantly inhibit mammary tumour formation in a 7,12-dimethylbenz[a]anthracene model of breast cancer in Sprague–Dawley rats  has also been shown to inhibit the production of lung metastases in an orthotopic model of renal cell carcinoma  combined with the conventional cytotoxic agent paclitaxel (20 mg kg−1 dose−1) produced a greater inhibition of tumour growth than that seen with either agent alone  inhibitor, in human xenograft models of lung and colon cancer . Further mutational analysis of both EGFR and RET may be necessary to better understand the clinical significance of this phenomenon.Achieving an optimal clinical response to ZD6474 may depend on several factors. These include its use in combination with other anticancer strategies and the stage of tumour development when treatment is initiated. RET gene can be activated by somatic rearrangements in papillary thyroid carcinoma or by point mutations in sporadic and familial (MEN2) medullary thyroid carcinomas. Therefore, ZD6474 might be especially beneficial in the treatment of thyroid tumours with known mutations in the RET gene (The activity of ZD6474 against RET kinase provides an opportunity to target specific patient subsets who may obtain particular benefit from this pharmacological activity. The In conclusion, ZD6474 is a potent, orally bioavailable inhibitor of VEGFR and EGFR tyrosine kinase activity. It is a novel molecular-targeted agent that has shown promising antitumour activity in preclinical models. Given early reports of clinical activity with this agent in NSCLC (


In addition, knockout murine models point to an essential role for AM in embryonic vasculogenesis and ischaemic revascularisation. AM expression is upregulated by hypoxia  and a potential role as a regulator of carcinogenesis and tumour progression has been proposed based on studies in vitro and in animal models. Nevertheless, translational research on AM, and in particular, confirmation of its importance in the vascularisation of human tumours has lagged behind. In this commentary, we review current progress and potential directions for future research into the role of AM in tumour angiogenesis.The angiogenic activity of peptide adrenomedullin (AM) was first shown in 1998 . Since then, a number of reports have confirmed the ability of AM to induce the growth and migration of isolated vascular endothelial and smooth muscle cells  Andrenomedullin belongs to the calcitonin gene peptide superfamily based on its homology with calcitonin gene-related peptide (CGRP) and amylin  has been described and characterised as human complement factor H (reviewed by N-terminus (∼100 amino acids), a single transmembrane domain, and a short intracellular domain (10 amino acids). Formation of heterodimers between RAMPs and CL is essential for proper cell surface targeting and to define ligand-binding selectivity of this GPCR (AM mediates its activities via heterodimeric receptors that are composed of a seven transmembrane (7TM) G-protein-coupled receptor (GPCR) calcitonin-receptor-like receptor , mitogen-activated protein kinase (MAPK) and focal adhesion kinase (p125FAK) plays a role in AM-induced angiogenesis . Thus, Ain vivo contributes to recruitment of VSMC to a newly formed capillary network  compared to controls . Adrenom22–52 and CGRP8–37, and small nonpeptide molecules), or (3) inhibit specific AM-induced intracellular signalling pathways   modulate AM expression (AM mRNA rybozyme), (2) alter AM binding to its receptor  . Severalin vitro and murine xenografted tumour models, studies in man have been limited to use of cancer cell lines and a few tumours.Although the role of AM has been studied using P=0.002), as measured by radioimmunoassay . Information on the distribution, regulation and function of endogenous AM receptors is poor, and data on their role in human tumour biology is not available. CL mRNA might be upregulated in some tumour types .in vitro studies have demonstrated that hypoxia upregulates AM expression in tumour cells and this needs to be validated in vivo by, for example, in situ hybridisation  to be widely expressed in normal adult tissue. Expression is greatest in the placenta and endometrium that are undergoing active angiogenesis, but was also significant in the adrenal medulla, lung and kidney (There is a need to identify the tumour types where expression of AM and its receptors correlate with, for example, vascularisation, prognosis and resistance to chemotherapy. This would single out tumours in which AM plays a role and where anti-AM therapeutic intervention may have potential. That antibodies to AM reduce tumour growth in animal models was a significant observation reminiscent of similar observations with anti-VEGF antibodies a decade earlier. The VEGF studies eventually led to the development of the anti-VEGF drug ‘Avastin’ and its efficacy in the treatment of advanced metastatic colon cancer (


The blastogenic response to autologous blood lymphocytes to whole-cell suspensions and to homogenates obtained from malignant lymphoma tissue has been investigated. Spleens were obtained from patients in whom laparotomy was performed for staging of malignant lymphoma. Cell suspensions prepared from tumour nodules were treated with mitomycin C and allowed to react with separated autologous blood lymphocytes for 6 days. Lymphocyte stimulation was measured by liquid scintillation counting after exposure to 3H-TdR. Cultures were also prepared in which autologous lymphocytes were treated with spleen tumour homogenate. Control experiments used spleens from staging procedures in which no tumour deposits were present, and normal spleens removed incidentally during other operations. In the controls, the uptake of TdR was never more than twice that of unstimulated lymphocytes. Greater degrees of lymphocyte stimulation were seen in 6 out of 14 patients, using whole tumour cells, and in 7 out of 16 patients, using tumour homogenates. The results indicate an antigenic difference between tumour and host cells, and suggest that lymphocytes can react to a tumour-associated antigen.


Loss of heterozygosity (LOH) frequently occurs in squamous cell carcinomas of the uterine cervix and indicates the probable sites of tumour-suppressor genes that play a role in the development of this tumour. To define the localization of these tumour-suppressor genes, we studied loss of heterozygosity in 64 invasive cervical carcinomas (stage IB and IIA) using the polymerase chain reaction with 24 primers for polymorphic repeats of known chromosomal localization. Chromosomes 3, 11, 13, 16 and 17, in particular, were studied. LOH was frequently found on chromosome 11, in particular at 11q22 (46%) and 11q23.3 (43%). LOH on chromosome 11p was not frequent. On chromosome 17p13.3, a marker (D17S513) distal to p53 showed 38% LOH, whereas p53 itself showed only 20% LOH. On the short arm of chromosome 3, LOH was frequently found (41%) at 3p21.1. The beta-catenin gene is located in this chromosomal region. Therefore, expression of beta-catenin protein was studied in 39 cases using immunohistochemistry. Staining of beta-catenin at the plasma membrane of tumour cells was present in 38 cases and completely absent in only one case. The tumour-suppressor gene on chromosome 3p21.1 may be beta-catenin in this one case, but (an)other tumour-suppressor gene(s) must also be present in this region. For the other chromosomes studied, 13q (BRCA-2) and 16q (E-cadherin), only sporadic losses (< 15% of cases) were found. Expression of E-cadherin was found in all of 37 cases but in six cases the staining was very weak. No correlation was found between clinical and histological parameters and losses on chromosome 3p, 11q and 17p. In addition to LOH, microsatellite instability was found in one tumour for almost all loci and in eight tumours for one to three loci. In conclusion, we have identified three loci with frequent LOH, which may harbour new tumour-suppressor genes, and found microsatellite instability in 14% of cervical carcinomas.


Varicella-Zoster virus causes chickenpox upon primary infection and shingles after reactivation. Currently available serological tests to detect VZV-specific antibodies are exclusively based on antigens derived from VZV-infected cells.We present a systematic approach for the identification of novel, serologically reactive VZV antigens. Therefore, all VZV open reading frames were cloned into a bacterial expression vector and checked for small scale recombinant protein expression. Serum profiling experiments using purified VZV proteins and clinically defined sera in a microarray revealed 5 putative antigens . These were rearranged in line format and validated with pre-characterized sera.The line assay confirmed the seroreactivity of the identified antigens and revealed its suitability for VZV serodiagnostics comparable to commercially available VZV-ELISA. Recombinant ORF68 (gE) proved to be an antigen for high-confidence determination of VZV serostatus. Furthermore, our data suggest that a serological differentiation between chickenpox and herpes zoster may be possible by analysis of the IgM-portfolio against individual viral antigens. The Varicella-Zoster virus (VZV) is a member of the neurotropic alphaherpesvirus subfamily of the Herpesviridae. VZV causes varicella (chickenpox) during primary infection and may cause herpes zoster (shingles) as secondary disease after reactivation from latency.Varicella can be considered as a harmless childhood disease. However, severe outcomes in the elderly, immunocompromised or resulting from congenital infection of the fetus or newborn are dreaded . A live One major goal of VZV-specific laboratory diagnosis is the identification of immunological markers that correlate with protection against varicella. Such markers are extraordinarily important, since the diagnosis of susceptibility to the disease implies therapeutic consequences as for example active or passive immunization within certain person groups . It is gE. coli Rosetta (DE3) revealed, that ~35.2% of all VZV proteins could be expressed recombinantly, and ~25.4% of all VZV proteins could be purified in 96 well format. When we performed serum profiling experiments with clinically defined VZV patient sera in a microarray format, ~27.8% of the purified VZV proteins could be identified as putative serological marker antigens. The respective recombinant antigens were purified in large scale as described by Soutscheck et al. . The latter vector has been constructed by insertion of a customized cassette consisting of 5'-NheI-HindIII-ATG-[RGS-His-tag]-EcoRV-[ccdB/CmR(rfB)]-EcoRV-XbaI-SalI-3' into the backbone of the bacterial expression vector pET-22b(+) . LR clonase reactions were incubated at 37°C for 2 h and subsequently transformed into chemically competent E. coli DH5α. Plasmid DNA of individual colonies grown on LB-plates supplemented with 100 μg/ml ampicillin  was isolated as described above and the integrity of the resulting pETG-A-His-N-VZV-ORF vectors was verified by HindIII/XbaI  restriction analysis.LR recombination reactions using LR-clonase II enzyme mix  were performed according to the manufacturers' instructions. Briefly, pENTR207-VZV-ORF vectors containing VZV-ORFs flanked by E. coli Rosetta (DE3). Transformed bacteria were selected on LB-plates supplemented with 100 μg/ml ampicillin . Pools of ten colonies per LB-plate were picked and resuspended in freezing media (LB-media supplemented with 15% glycerol). The respective 71 pools of transformed bacteria were stored in a 96-well (round bottom) plate  at -80°C until further analysis.Systematic small scale protein expression and purification was performed as described recently  with min96 manual 96-channel pipetting tool . The 96-well glycerol culture plate was thawed and used for inoculation of 1.5 ml LB-media supplemented with 100 μg/ml ampicillin  in a 96 (round bottom) deep well block . The latter block was incubated in a bacterial shaker at 37°C over night. For induction of recombinant protein expression, 1.4 ml LB-media were inoculated in a fresh 96 deep well block with 100 μl of the latter over night culture and incubated in a bacterial shaker for 3 h at 37°C before addition of IPTG to a final concentration of 1 mM and an additional shaking for 6 h at 37°C. The induced bacteria were pelleted after centrifugation of the 96 deep well block at 2.000 g, resuspended in 100 μl lysis buffer , and subsequently cleared by utilizing a Millipore 96-well filtration (clearing) plate  according to the manufacturers' instructions. The cleared bacterial lysates were analyzed by SDS/PAGE followed by Western blotting using the monoclonal mouse anti RGS-His antibody  for the presence of recombinant, His-tagged proteins.For analysis of recombinant protein expression, all steps were performed in 96-well format using a LiquidatorFor fast small scale recombinant protein purification, the bacterial pools that have been detected positive for the presence of His-tagged VZV-proteins were induced in individual 5 ml batch cultures for 6 h at 37°C. Protein purification under denaturating conditions was performed using Ni-NTA spin columns  according to the manufacturers' instructions. Protein purification was verified by SDS/PAGE following Coomassie staining. Purified His-tagged proteins were stored at -20°C until further usage.recomDot, Mikrogen, Germany) were processed according to the manufacturer's instructions. Briefly, purified recombinant proteins and VZV lysate  as a control were spotted on nitrocellulose microarrays with a capacity for 34 spots . All antigens were spotted in duplicate. Arrays were incubated with 2 ml diluted serum . For detection of specifically bound antibodies anti-human IgG- and anti-human IgM-peroxidase conjugates  were used. Visualization of immune complexes was done with tetramethylbenzidine (TMB) colorization substrate . In order to identify putative serological VZV candidate antigens, the microarrays were probed with different clinically and serologically defined patient sera. Clinically defined serum samples were derived from patients suffering from acute varicella or zoster. Serologically defined serum samples (e.g. VZV-IgG negative samples) were pre-analyzed for the presence of VZV-IgG by the commercially available Enzygnost wcELISA . All clinically defined serum samples were additionally chracterized for their VZV-IgG and VZV-IgM titers by Enzygnost wcELISA .For serum profiling experiments, the small scale purified, His-tagged VZV proteins were spotted on microarrays. Microarrays  was performed as described previously ,33. The d before .Individual dilutions of the purified recombinant antigens were applied directly onto nitrocellulose membranes in different lines. The appropriate line conditions for all recombinant antigens were determined empirically with standard serum samples. Membranes were blocked with 1% skim milk solution in phosphate-buffered saline, air dried, and cut into individual test strips. Strips were stored at 4°C. Processing of nitrocellulose test strips was performed following the instruction manual for recomLine EBV  using the reagents supplied in the kit. Briefly, serum samples were applied at 1:100 dilutions and incubated together with the nitrocellulose test strips for 1 h at room temperature. Following three washing steps of 5 min each, a second incubation of 45 min with peroxidase-labelled secondary antibody (anti human IgG or IgM) was performed. Strips were stained for about 8 min using tetramethylbenzidine after three additional washing steps of 5 min each. Controls were used as described previously ,33. The MGVP, TJ, AL, JH, HN, GJ and AB have non-financial competing interests.KIP, CN and MS have received salaries from Mikrogen GmBH.MGVP carried out the recombinatorial cloning, sequence analysis, expression and purification of proteins in small format, ELISAs assays, data analysis and wrote the manuscript. KIP carried out large scale protein purification, microarray screen, line development and data analysis, participated in the coordination of the study and help to draft the manuscript. TJ, CN and MS carried out large scale protein purification, microarray screen, line development and validation of VZV antigens in Line format. AL, JH, HN and GJ participated in the design of the study. AB conceived the study, and participated in its design and coordination and wrote the manuscript. All authors read and approved the final manuscript.Systematic pipeline for the identification of novel serological markers of VZV infection. A) Nested PCR for the amplification of VZV ORFs with attB sites, B) BP reaction into pDONR207, C) Characterization of resulting pENTR207 vectors by BanII restriction analysis and sequencing, D) LR reaction to insert the customized bacterial expression vector pETG-A-His-N- [rfB], E) Characterization of resulting pETG-A-His-N-VZV-ORF vectors by HindIII and XbaI restriction analysis and sequencing, F) Transfection of expression vectors into E. coli Rosetta (DE3) and induction of protein expression with IPTG, G) Analysis of protein expression by Western blotting using an anti-RGS-His antibody, H) Analysis of purified proteins by SDS-PAGE and Coomassie staining, I) Screening for serologically reactive antigens in microarray format, J) Rearrangement of screened marker antigens in Line format.Click here for fileValidation of the VZV RecomLine with clinically defined serum samples. This table depicts all serological parameters of the clinically defined patient sera.VZV-IgG and IgM ELISA titres were assayed by wcELISA . Qualitative RecomLine VZV IgG and IgM reactivities towards individual antigens are depicted as "1" (reactive) and "0" (non reactive).Click here for fileAnalysis of possible crossreactivities of the RecomLine VZV recombinant antigens with serum samples of defined antibody status to HSV. This table depicts the cross-reactivity of all serologically defined VZV-IgG negative patient samples according to their HSV (IgG) status. No cross-reactivity in the in the RecomLine VZV IgM assay can be observed. The recomLine VZV IgG assay exhibits some reactivities against ORFs 4, 14, 49 but not against ORF68. No correlation of cross-reactivities and HSV-1 status can be observed.Click here for file


Cell survival was investigated in an intraocular retinoblastoma-like tumour 30 min to 48 h after photodynamic therapy. The survival of the cells was assessed by an in vivo to in vitro colony forming assay, estimated by either the plating efficiency of the treated tumour cells compared to non-treated cells or the number of clonogenic cells per mg excised tumour. Curves showing cell survival as a function of the time between light irradiation and excision of the intraocular tumours were biphasic. This suggests more than one PDT tissue destruction mechanism in vivo (i.e. an early direct cell damage plus a subsequent late damage occurring in the tumour tissue left in situ after treatment). The delayed mechanism may be due to changes in the environment of the tumours probably caused by vascular damage. Tumour cells sensitised by Photofrin II in vivo and excised from the eyes were damaged by light when irradiated in vitro and this was dependent on the light energy dose. This showed that cellular Photofrin II uptake in the eye tumours was sufficient for direct cell damage and thus supports the suggestion that direct and indirect tumour destruction occurs in this eye tumour after photodynamic therapy.


Recent data have shown that the in vitro and in vivo cytotoxicity of bioreductive drugs could be significantly increased when combined with chemotherapy drugs such as cisplatinum, depending on the timing of administration. The aim of this study was to define the toxicity  and the activity  of a bioreductive drug, tirapazamine, alone and combined with chemotherapy agents  on nude mice bearing xenografted human tumours: a rectal carcinoma (HRT18) and a melanoma (Na11+). Animal lethality was markedly increased when tirapazamine at the lethal dose 10% was combined with the other drugs. For the HRT18 tumour the combination of tirapazamine and bleomycin significantly increased the delay of regrowth compared with bleomycin alone (P = 0.04) and was more cytotoxic than tirapazamine alone (P = 0.04). For the Na11+ tumours the combination of tirapazamine with VP16 significantly increased tumour doubling time compared with the controls (P = 0.001) or VP16 alone. The combination of tirapazamine and VP16 was more cytotoxic than VP16 alone (P = 0.0001). When compared with c-DDP or tirapazamine alone, there was a significant decrease in plating efficiency when tirapazamine and c-DDP were given at the same time (P = 0.04), but not when tirapazamine was given 3 h before c-DDP. In conclusion, tirapazamine was shown to be cytotoxic against clonogenic human tumour cells. Its efficacy in vivo may depend on its combination with already active chemotherapy drugs on the tumour model used. The timing of administration may be less important than previously thought.


Fluid transport parameters in intracranial tumours influence the delivery of therapeutic agents and the resolution of peritumoral oedema. The tumour and cortex interstitial fluid pressure (IFP) and the cerebrospinal fluid pressure (CSFP) were measured during the growth of brain and pial surface tumours [R3230AC mammary adenocarcinoma (R3230AC) and F98 glioma (F98)] in rats. Intratumoral and intracranial pressures were also measured in rodents and patients treated with dexamethasone, mannitol and furosemide (DMF), and hypocapnia. The results show that (1) for the R3230AC on the pial surface, IFP increased with tumour volume and CSFP increased exponentially for tumours occupying a brain volume of 5% or greater; (2) in F98 with volumes of approximately 10 mm3, IFP decreased from the tumour to the cortex, whereas for tumour volumes > 16 mm3 IFP equilibrates between F98 and the cortex; (3) DMF treatment reduced the IFP of intraparenchymal tumours significantly and induced a pressure gradient from the tumour to the cortex; and (4) in 11 patients with intracranial tumours, the mean IFP was 2.0 +/- 2.5 mmHg. In conclusion, the IFP gradient between intraparenchymal tumours and the cortex decreases with tumour growth, and treatment with DMF can increase the pressure difference between the tumour and surrounding brain. The results also suggest that antioedema therapy in patients with brain tumours is responsible in part for the low tumour IFP.


We compared para-, meta- and ortho-isomers of meso-tetra(hydroxyphenyl)porphyrin  and the potassium salt of the para compound (K-p-THPP) with haematoporphyrin derivative (HpD) and Photofrin II in their ability to sensitise tumours, skin and brain to light. HpD and Photofrin II induced modest tumour photosensitisation at the cost of substantial skin and brain sensitisation. At doses low enough to keep sensitisation of these normal tissues within acceptable limits, tumour sensitisation was sufficient to give necrosis only approximately 2 mm deep after exposure to 10 J cm-2 light. In contrast, doses of p-THPP, K-p-THPP and m-THPP that produced skin and brain sensitivity within acceptable limits sensitised tumours enough to give 4-9 mm necrosis after 10 J cm-2 light. m-THPP was, on a molar basis, about 25-30 times as potent as HpD and Photofrin II in sensitising tumours. o-THPP was also a potent tumour photosensitiser, but induced a prohibitive degree of skin photosensitivity even at low doses. It is unlikely that these differences in relative selectivity are due to differences in such photophysical parameters as optimum activating wavelength (which would affect tissue penetration by light), or light absorption, and physicochemical factors that determine tissue localisation may be involved. The high tumour sensitising potency and favourable tissue selectivity of m-THPP, p-THPP and K-p-THPP make them promising candidates for clinical tumour phototherapy.


Macrophages have been isolated from ascitic and collagenase-dispersed tumours from patients undergoing surgery for ovarian cancer. Macrophages were present in varying proportions in both sites, though the ration of macrophages to tumour cells was higher in ascites. Marked variation in size (as detected by sedimentation velocity) and cytochemical markers in the macrophages was noted. Highly enriched macrophage fractions were isolated from the ascites and collagenase-dispersed solid tumours by a combination of sedimentation velocity and selective EA RFC or adherence techniques. Suppressor activity in the PHA assay was detected in tumour macrophages (4/10 giving less than 50% inhibition), ascitic macrophages (1/15) and blood monocytes (2/7). Lymphocyte fractions from tumours were unresponsive to PHA and failed to suppress the blood response. Suppressor activity was also present in the purified tumour-cell fraction of 6/14 patients. ADCC activity was tested in a few patients. When the activity was determined against the SB target cells, tumour-derived macrophages were inactive, whereas the ascitic fraction showed low but significant activity which averaged much lower than patients blood values. The ADCC assays carried out with the CRC target cell indicated activity within the range of patient blood values in 4/4 ascites and 2/4 tumour macrophage fractions. Cytotoxicity was also assessed against co-purified autologous tumour cells. Although activity was detected in many of the tests, the results seemed to reflect target cell sensitivity. There appeared to be a correlation between cytotoxicity with test macrophages and normal blood mononuclear cells. The results indicate that the cytochemical heterogeneity and the variation in size between macrophage fractions is associated with a spectrum of activities.


We have employed molecular probes produced from DNA fragments of human papillomavirus, cloned into prokaryotic vectors, to detect virus nucleic acid sequences in extracts of human oral tissues. The study was conducted with duplicate coded snap-frozen tissue biopsies from which frozen sections had been taken to accurately assess the pathology of each particular sample. The results show that a large proportion of the oral biopsies contained DNA which hybridized to the viral DNA probes, even under conditions of high stringency. The presence of virus did not correlate with neoplasia in the tissues examined, but HPV like sequences were found in a high proportion (80%) of biopsies taken from areas of keratosis and lichen planus and also in 41 to 46% of normal and tumour tissues.


Male C57BL/6 mice were challenged s.c. with 2 × 106 viable Sarcoma-180 tumour cells and evaluated sequentially in parallel with saline-injected controls over a 21-day experimental period. Before challenge, immunoreactive plasma fibronectin was 1050-1150 μg/ml. Minimal tumour growth occurred until 6 days after tumour challenge. There was then a rapid increase in primary tumour size, especially over the 7-14-day interval, with a plateau of growth over the 18-21-day interval. Immunoreactive plasma fibronectin was significantly (P < 0·05) raised at 3 and 7 days after tumour challenge. A rapid rise (P < 0·001) to 2816·6 ± 158·9 μg/ml was observed at 14 days followed by a modest decline at 21 days. Bioassayable opsonic activity increased (P < 0·5) with the rise in immunoreactive fibronectin 3 and 7 days after tumour challenge, but the rapid rise in immunoreactive fibronectin over the 7-14-day interval was associated with a significant (P < 0·5) fall in bioassayable opsonic activity. Thus, the rapid rise in immunoreactive plasma fibronectin parallels the rapid rate of tumour growth, but is associated with a fall in opsonically active plasma fibronectin. Dissociation between immunoreactive and opsonically active plasma fibronectin may be mediated by inhibition and/or alteration of circulating fibronectin during rapid tumour growth. Alternatively, it may reflect increased release of antigenically related protein (i.e. cell-surface fibronectin) during rapid tumour growth, which may have limited biological opsonic activity.Immunoreactive and bioassayable plasma fibronectin (opsonic α


Previously we reported that fusogenic liposomes, prepared by fusing simple liposomes with Sendai virus particles, could introduce their contents directly and efficiently into the cytoplasm. In this study, we examined the anti-tumour activity of fusogenic liposomes containing fragment A of diphtheria toxin (DTA). Fusogenic liposomes containing DTA showed high cytotoxicity against sarcoma-180 (S-180) cells in vitro. When these liposomes were administered into the abdominal cavity of ddY mice carrying S-180, tumour cells completely disappeared in four of six tumour-bearing mice without decrease in body weight. Neither simple liposomes containing DTA nor empty fusogenic liposomes had any effect on tumour suppression. We conclude that fusogenic liposomes containing DTA are new and potentially effective tools for the treatment of ascites tumours without any severe side-effects.


Alternatively, experimental metastases were established by tail-vein injection of 50 000 4T1 cells. Mice received either the selective COX-2 inhibitor, SC-236 or drug vehicle for 14 days (n=12 per group). SC-236 treatment significantly reduced tumour burden, the number and size of spontaneous metastases following primary tumour excision. SC-236 treatment also reduced tumour burden, the number and size of experimental metastases. Immunohistochemical staining demonstrated that COX-2 inhibition reduced microvessel density and increased apoptosis within both spontaneous and experimental metastases. These data clearly demonstrate that the selective COX-2 inhibitor, SC-236, has potent antimetastatic activity against both spontaneous metastases arising following primary tumour excision and experimental metastases.Cyclooxygenase-2 (COX-2) expression is increased in breast cancer and surgery has been shown to increase the growth of metastatic tumours. We investigated the effect of selective COX-2 inhibition on the growth of metastases in either an experimental metastasis model or following excision of a murine primary breast tumour. 50 000 4T1 mammary carcinoma cells were injected into the mammary fat pad of female BALB/c mice. When the mean TD reached 8±0.4 mm, tumours were excised and the mice were randomised into two groups ( In most cancer patients, metastases are already present at time of diagnosis  and whilSeveral studies show that regularly taking aspirin or other conventional nonsteroidal anti-inflammatory drugs (NSAIDs) provides a 40–50% reduction in the relative risk of death by colon cancer, indicating that inhibition of cyclooxygenase (COX), both COX-1 and COX-2, has a chemopreventive effect . Selecti2 (PGE2), is produced in large amounts by some tumours and has been implicated in the promotion and growth of malignant tumours  and water.Female 10–12-week-old BALB/c mice  were used. The animals were acclimatised for 1 week and caged in groups of five or less in an air-conditioned room at ambient temperature of 21–22°C and 50% humidity under a 12-h light–dark cycle (lights at 0800). Animals were housed in a licensed biomedical facility and all procedures were carried out under animal license guidelines of the Department of Health, Ireland and in accordance with the UK Co-ordinating Committee on Cancer Research (UKCCCR) Guidelines for the Welfare of Animals in Experimental Neoplasia . Cell cultures were incubated in an atmosphere of 5% CO2 in air at 37°C. Tumour cells were harvested from subconfluent cultures with 0.25% trypsin-0.02% EDTA. Trypsin was neutralised with medium containing 10% FBS, washed three times in phosphate-buffered saline (PBS) and resuspended in PBS at 5 × 105 ml−1 for injection. Only single cell suspensions of greater than 90% viability as determined by trypan blue exclusion were used for injections.4T1 tumour cells, a spontaneously metastasising mammary adenocarcinoma cell line were a generous gift from Dr Fred Miller, Duke University. Cells were maintained as monolayer cultures in Dulbecco's modified Eagle medium supplemented with 10% foetal bovine serum (FBS), sodium pyruvate and 4) were injected into the mammary fat pad behind the left forefoot after anaesthesia was induced and maintained with inhalational halothane. Primary tumours were measured on alternate days following injection of tumour cells using a Vernier calipers. Tumour diameter (TD) was calculated as the square root of the product of two perpendicular diameters (n=12 per group). The treatment and control groups received daily intraperitoneal injections of either 200 μl of the selective COX-2 inhibitor, SC-236 (6 mg kg−1 in 1% v v−1 dimethylsulphoxide DMSO) or vehicle (1% DMSO), respectively, for 14 days. Animals were killed and lungs excised. Lungs from seven mice per group were fixed in Bouins solution and the number and size of lung lesions determined with the aid of a dissecting microscope (4T1 cells (5 × 10iameters . When men=12 per group) received 5 × 104 4T1 cells via tail-vein injection. Controls received daily intraperitoneal injections of 200 μl drug vehicle (1% DMSO) while the treatment group received SC-236 by intraperitoneal injection (6 mg kg−1 in 1% DMSO) starting on the day of tumour cell injection. After 14 days, mice were killed, lungs removed, fixed in Bouins solution and the number and size of lung lesions determined as above (n=7). Lungs from remaining five mice per group were processed for immunohistochemistry. All experiments were performed in duplicate.For studies on experimental metastases BALB/c mice  according to the manufacturer's instructions .At the time of killing, animals were anaesthesised with halothane and their chests cleaned with ethanol. Blood was obtained via a closed cardiac puncture by means of a 22-gauge hypodermic needle and a subxiphoid approach. Blood was allowed to clot for 2 h at room temperature and centrifuged for 20 min at 1100 n=5) were embedded in OCT compound , snap frozen in liquid nitrogen and stored at −80°C. Microvessel density was determined as described previously by staining with MECA 32 antibody ) (Harvested lungs (μm) were fixed in cold acetone for 5 min, 1 : 1 acetone : chloroform for 5 min, acetone for 5 min and washed 3 times in phosphate-buffered saline (PBS) for 5 min each. Apoptotic cells within tumour metastases were stained using the in situ cell death detection kit according to the manufacturer's instructions . Peroxidase activity was visualised by the precipitation of 3,3′-diaminobenzidene (DAB) and sections were lightly counterstained with haematoxylin. Apoptotic cells stained brown against a blue background. Only cells which stained brown and had the morphological appearance of an apoptotic cell were counted as apoptotic cells, necrotic cells were easily distinguished and were excluded. Apoptosis was expressed as the number of apoptotic cells in three high-power fields per section (× 400 magnification (× 40 objective and × 10 ocular)). Lung sections from each of five mice per group were analysed. Data are expressed as mean±s.e.m.Harvested lungs were embedded in OCT compound , snap frozen in liquid nitrogen and stored at −80°C. Sections (8 t-test using Instat for Windows statistics package (Graphpad Software Inc). Data were taken as significant where P<0.05.Data are expressed as mean±standard error mean (s.e.m.). Differences between treatment groups were determined by unpaired in situ (We previously demonstrated that COX-2 inhibition decreased the number of metastases from a primary mammary fat pad 4T1 tumour P=0.04). The lung nodules in the control group were also significantly larger than those in the SC-236-treated mice (median diameter 0.98 (0.81–1.1) mm vs 0.76 (0.64–0.87) mm, P=0.002). Excised lungs were weighed and metastatic burden expressed as lung-to-body ratio as before (P=0.01).4T1 mammary adenocarcinoma cells spontaneously metastasise to the lungs from the mammary fat pad . MammaryTable 1vs 97 (67–135) (P=0.02). Furthermore, the size of metatases in the control group were also significantly larger than those in the SC-236-treated mice (median diameter 0.77 (0.33–0.99) vs 0.65 (0.18–0.79) mm, P=0.046).  P=0.006) (P=0.006)  . This inP=0.006) .P<0.05) (−1) relative to controls (83.5±20.9 pg ml−1) (P=0.02). The antiangiogenic effect of SC-236 treatment was also evident in the experimental metastasis model (12.8±1.8 vessels/h.p.f. in SC-236 treated mice compared to 22.7±2.9 vessels/h.p.f. in controls. (P=0.017). vs 93.2±55.3 pg ml−1) (P=0.017).To further investigate the mechanisms by which the selective COX-2 inhibitor (SC-236) reduced metastasis, we examined the effect of treatment on angiogenesis within the pulmonary metastases following excision of primary tumour. Vascularisation was identified by staining tumours with MECA-32 antibody  . The redWhile surgery results in the successful removal of the primary tumour in the majority of patients, a significant number of patients subsequently die of metastatic disease. The release of proangiogenic factors in the postoperative period may contribute to the growth of previously dormant metastases. In addition, removal of the primary tumour, a source of the endogenous angiogenesis inhibitor, angiostatin, may remove a ‘brake’ on metastatic growth  mice when compared to tumours grown in wild-type mice. Here, we show that COX-2 inhibition also decreases angiogenesis within spontaneous and experimental metastases suggesting that in addition to increasing tumour cell apoptosis these drugs exert their antimetastatic effect by also reducing angiogenesis in the metastases. Our hypothesis is further supported by studies indicating a role for COX-2 in the production of angiogenic factors by cancer cells (Microvessel density within the primary tumour, a measure of the degree of angiogenesis, is an independent predictor of metastatic disease in breast cancer patients ((in vivo . Williamer cells , increas−/−, COX-1−/−, or COX-1−/−/COX-2−/− mice (κB signalling pathway (α, δ and γ have been identified in humans, each with a different pattern of expression. Fatty acids and certain fatty acid metabolites have been suggested as endogenous ligands for these receptors. Indomethacin can bind to, and induce transcriptional activity of PPARα and γ isoforms, and PPARδ is a target for sulindac (L (in vitro is in the 50–100 μM range (In addition to inhibiting tumour growth via COX inhibition, it is possible that COX-2 inhibitors may also effect other target molecules. There are studies which show that NSAIDs can have COX-independent activity. First, there is no correlation between sensitivity to NSAID-induced cell death and levels of COX expression in transformed fibroblasts derived from COX-2 pathway . PeroxisμM range .In summary, we have clearly demonstrated that COX-2 inhibitors have a potent antimetastatic effect increasing apoptosis and decreasing angiogenesis within both spontaneous and experimental metastases. The selective COX-2 inhibitors are in everyday clinical use, cheap and with a known side effect-profile. Our data suggest that COX-2 inhibitors may have clinical utility in the management of metastatic disease especially in the perioperative period.


Mixed epithelial and stromal tumour (MEST) represents a recently described benign composite neoplasm of the kidney, which predominantly affects perimenopausal females. Most tumours are benign, although rare malignant cases have been observed.A 47-year-old postmenopausal female presented to the urologist with flank pain. A CT scan of the abdomen showed a 30-mm-in-diameter uniform mass adjacent to the pelvis of the left kidney. Surgical exploration showed a tumour arising from the lower anterior hilus of the left kidney. The tumour could be excised by preserving the kidney. By intraoperative frozen section the tumour showed characteristic features of MEST with epithelial-covered cysts embedded in an "ovarian-like" stroma. Additional immunohistochemistry investigations showed expression for hormone receptors by the stromal component of the tumour.MEST typically presents in perimenopausal women as a primarily cystic mass. Commonly, the tumour arises from the renal parenchyma or pelvis. The tumour is composed of an admixture of cystic and sometimes more solid areas. The stromal cells typically demonstrate an ovarian-type stroma showing expression for the estrogen and progesterone receptors.MEST represents a distinctive benign tumour entity of the kidney, which affects perimenopausal woman. The tumour should be distinguished from other cystic renal neoplasms. By imaging studies it is difficult to distinguish between a benign or malignant nature of the tumour. Thus, intraoperative frozen section is necessary for conservative surgery, since the overall prognosis is favourable and renal function can be preserved in most cases. The mixed epithelial and stromal tumour (MEST) of the kidney represents a recently described benign tumour, which has to be distinguished from other renal neoplasms. The term MEST was first introduced by Michal and Syucek in 1998-4.We report the unusual case of a MEST arising from the left kidney as a cystic tumour in an exophytic fashion. Since the diagnosis was secured by intraoperative frozen section, tumour excision without nephrectomy could be performed as treatment of choice.A 47-year-old postmenopausal female patient presented to us with increasing left-sided flank pain. Physical examination was unremarkable. Aside from a well-medicinally treated hypertension no accompanying disease was present. Her routine blood investigations were normal. Urine cytology revealed suspicions of atypical urothelial cell elements. Retrograde ureterography showed an obstruction of the proximal left ureter due to a tumour projecting to the lower-left renal pelvis Figure . A contrOn surgical exploration there was a well-circumscribed tumour mass arising from the lower anterior pole of the left kidney attached with a narrow pedicle to the papilla of the lower chalice group Figure . The tumGross examination showed a 2.5 × 2 × 1.5-cm-in-diameter, well-encapsulated partly cystic tumour with a 2-cm-long narrow pedicle. Histologically, the tumour was composed of cysts embedded in a collagenous stroma containing bundles of spindle cell proliferations with variable cellularity. The cysts varied significantly in diameter and were covered by a flat or cuboidal epithelium with areas of clear cell differentiation and hobnail appearance. The thickness of the cyst septi ranged from one to 6 mm microscopically. The relative proportion of the stromal component to the entire tumor (stroma to cyst ratio) was about 55%. No dysplasia, increased mitotic activity or tumour necrosis as signs for malignant transformation were observed. A distinctive feature was focal condensation of stromal cell adjacent to the cysts Figure  and 3b. Mixed epithelial and stromal tumour (MEST) represents a recently described tumour entity of the kidney of unknown etiology ,5,6. In The patients usually present to the urologist with non-specific symptoms, such as flank pain, hematuria, or symptoms primarily suggestive of genitourinary infections. The mean tumour size at primary diagnosis is about 6 cm in diameter ,5,6. DifNearly all cases described so far demonstrated a benign course without tumour recurrence. However, four cases of aggressive MEST have been described in the literature so far -12. ThreThe differential diagnoses include renal tumours, which can show at least a partly cystic morphology, such as partially cystic differentiated nephroblastoma, multilocular cystic renal carcinoma, angiomyolipoma with epithelial cysts or in rare cases synovial sarcoma ,5,6.MEST demonstrate morphological overlap to cystic nephroma, with which it can be confused ,14. BothSince some tumours have gross and microscopic features intermediate between cystic nephroma and MEST, it is considered that both tumours might represent different morphological variants of the same tumour entity ,14,17,18Mixed epithelial and stromal tumour represents a distinctive benign tumour of the kidney that should be distinguished from other cystic renal neoplasms. Prognosis of this tumour is favourable in nearly all cases published so far. Only rare cases of malignant transformation have been published. In summary, MEST represents a benign mostly cystic tumour of the kidney, which is predominately observed in middle aged, perimenopausal women. Knowledge of this certain but rare tumour entity is important, since in most cases conservative surgery with preservation of kidney function is the therapy of choice.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.MR, JK and PM participated in conception of the idea and writing of the manuscript. WM and PM performed the histopathological interpretation of the tumour tissue.


In this paper we investigate the reactivity pattern of T cells from stomach carcinoma patients against autologous tumour cells. T cells obtained from the tumour environment, tumour-draining lymph nodes and peripheral blood were cloned in 78 patients with stomach cancer and anti-tumour cytotoxic T lymphocytes (CTLs) precursor frequencies were assessed in each sample by using limiting dilution analysis. When tumour-specific CTLs were tested for specific T-cell killing by using only low doses of Interleukin 2 (100 U ml-1), a moderate rate of proliferation frequency of T cells (0.047) and specific cytotoxicity (12%) were observed in lymph node populations. When both IL-2 and autologous tumour cells in mixed lymphocyte tumour cultures (MLTCs) were used for stimulation, a dramatic increase in number (0.1) and in specific lytic activity (46%) could be measured. No effect or specific activity to tumour cells was observed with peripheral blood lymphocytes and tumour-infiltrating lymphocytes.


People at risk from coronary heart disease and large bowel cancer are drawn from the same urbanised, industrialised Western populations. Whilst changes in blood lipids are well recognised in heart disease, little is known of their role in large bowel cancer. This study investigates serial alterations in blood lipids in the 1,2-dimethylhydrazine (DMH) rat model of colon cancer. Eighty Wistar rats received a 5 weekly regimen of DMH. At week 10, and at 5 weekly intervals until week 40, random groups of 10 rats were killed and blood taken for total and free cholesterol, phospholipids, triglycerides and liver enzymes. All colonic neoplasms were histologically classified either as adenomas or carcinomas with groups being allocated into tumour-free (n = 16) or tumour-bearing (n = 54), the latter group being further sub-divided into animals with adenoma alone (n = 8) and those with carcinoma (n = 46). Results were considered both sequentially and according to tumour status. Sequential results showed that with increase in colonic neoplasms with time there were accompanying increases in free and % free cholesterol and in phospholipids (P less than 0.001). There were no changes in total cholesterol, triglycerides or liver enzymes. Results according to tumour status showed that whilst there was no difference in total cholesterol or triglycerides between tumour-free and tumour-bearing rats, there was a significant increase in free (P less than 0.01) and % free cholesterol (P less than 0.001) and a decrease in phospholipids in the tumour-bearing animals (P less than 0.001). There was no difference in any serum lipid between tumour-free and adenoma-bearing rats. In animals with carcinoma, while there was no difference in total cholesterol or triglycerides, there was an increase in free (P less than 0.005) and % free cholesterol (P less than 0.001) and a decrease in phospholipids (P less than 0.001) compared to tumour-free rats. The data show for the first time a clear relationship between blood lipids and the presence or absence of large bowel cancer.


Finally, 131I-labelled antibody was used to investigate the potential of radioimmunotherapy in SW1222 tumours. Using immunohistochemistry, we confirmed extra domain B expression in the tumour vasculature. Immunofluorescence demonstrated localisation and retention of injected Cy3-labelled L19-SIP at the abluminal side of tumour vessels. Biodistribution studies using a 125I-labelled antibody showed selective tumour uptake in both models. Higher recorded values for localisation were found in the SW1222 tumours than in the LS174T (7.9 vs 6.6 %ID g−1), with comparable blood clearance for both models. Based on these results, a radioimmunotherapy study was performed in the SW1222 xenograft using 131I-Labelled L19-SIP (55.5 MBq), which showed selective tumour uptake, tumour growth inhibition and improved survival. Radio- and fluorescence-labelled L19-SIP showed selective localisation and retention at vessels of two colorectal xenografts. Furthermore, 131I-L19-SIP shows potential as a novel treatment of colorectal tumours, and provides the foundation to investigate combined therapies in the same tumour models.Angiogenesis is a characteristic feature of tumours and other disorders. The human monoclonal antibody L19- SIP targets the extra domain B of fibronectin, a marker of angiogenesis expressed in a range of tumours. The aim of this study was to investigate whole body distribution, tumour localisation and the potential of radioimmunotherapy with the L19-small immunoprotein (SIP) in colorectal tumours. Two colorectal tumour models with highly different morphologies, the SW1222 and LS174T xenografts, were used in this study. Localisation and retention of the L19-SIP antibody at tumour vessels was demonstrated using immunohistochemistry and Cy3-labelled L19-SIP. Whole body biodistribution studies in both tumour models were carried out with  Radioimmunotherapy (RIT), the targeting of radiation to the tumour using monoclonal antibodies against tumour-associated antigens, has been used as a form of cancer treatment in both animal xenograft models as well as in humans  and a small immunoprotein (SIP). The L19-SIP is an 80 kDa fragment composed of two scFv fused to the human ∈s2–CH4 domain to provide a covalent stabilisation of the dimer , using both radio- and fluorescence-labelled antibody. We demonstrate that the radiolabelled antibody localises selectively in both tumour models. Furthermore, by employing high-resolution multi-fluorescence microscopy, we have been able to show that the antibody localisation is specific to the abluminal surface of the vessel. Finally, we show that RIT with The L19-SIP (Mw 80 kDa), which targets the ED-B domain of FN consists of a fusion between two scFv and a CH4 domain of the human IgE, which mediates the homodimerisation of recombinant antibody . Biodistribution experiments were carried out when tumours reached approximately 1 cm3. For therapy experiments, mice with palpable tumours of approximately 0.1 cm3 were used. All animal experiments were in compliance with the UK Co-ordinating Committee on Cancer Research (UKCCCR) guidelines for the Welfare of Animals in Experimental Neoplasia.Passaging was by subcutaneous implantation of small tumour pieces  for 30 min, followed by a1 h incubation with 2 μg ml−1 L19-SIP antibody at room temperature. After washing with PBS, the sections were then incubated with a horseradish peroxidase conjugated goat anti-human antibody  for 1 h at room temperature, rinsed in PBS, and then incubated with the avidin–biotin complex  following the manufacturer's instructions. Sections were developed with 3,3-diaminobenzidine  and counterstained with haematoxylin. Finally, sections were mounted in dibutyl phthalate xylene and visualised under a light microscope.Immunohistochemistry was performed on 10 μg of labelled antibody was injected intravenously into the tail of the mouse 6 h before killing. Tumours were then excised, snap frozen in isopentane (cooled in liquid nitrogen), and stored at −80°C until sectioned.The L19-SIP antibody was labelled with Cy3-NHS  according to the manufacturer's instructions. The final antibody to dye ratio was 1 : 5. A total of 100 Perfusion: the in vivo DNA-binding dye Hoechst 33342 (10 mg kg−1) was injected intravenously 1 min before the mice were killed.Blood vessels: an anti-CD31 antibody was used to stain for blood vessel distribution, and the relevant immunohistochemical staining procedures were performed (see below).Nuclear staining: DAPI  was used as a nuclear marker. This produces a blue fluorescence when bound to DNA.To relate Cy3-L19-SIP antibody distribution to tumour morphology/pathophysiology, the following parameters were studied by multi-fluorescence microscopy:μm) were first fixed in acetone for 10 min at room temperature, and then incubated with a 1 : 2 dilution of unconjugated anti-CD31 rat anti-mouse antibody (a kind gift from Professor A Mantovani) for 1 h at room temperature. After rinsing in PBS, sections were incubated with a 1 : 200 dilution of an Alexa Fluor 488 goat anti-rat antibody  for 1 h at room temperature. After further rinsing with PBS, sections were mounted in either mounting medium containing DAPI (Vector Laboratories) or in PBS and viewed using an Axioskop 2 microscope , fitted with a computer-controlled motorised stage. Images were captured by an AxioCam digital colour camera using KS300 image analysis software   in 0.1 M phosphate buffer, pH 6.8; 50.18 MBq 125I (Amersham Biosciences). The reaction was stopped after 4 min by the addition of 100 μl of ascorbic acid . The solutions from two separate labellings were combined and purified on a PD-10 column and eluted with 0.9% NaCl/ascorbic acid . For the PD-10 column, the first 2.5 ml (1 ml sample volume and the first 1.5 ml) were discarded and the radiolabelled L19-SIP was collected in the next 2 ml.L19-SIP was labelled according to the IODO-GEN method as described by 131I-L19-SIP for therapy was performed according to the ‘IODO-GEN-coated MAb method’ (131I (444 MBq) was added to a glass vial in 1 ml NaOH (1 mmol l−1), after which 10 μl of ascorbic acid (1.4 mg ml−1) was added. After 5 min, 400 μl of phosphate buffer  was added, followed by 3 ml L19-SIP (approximately 1.5 mg) and 35 μl IODO-GEN solution (1 mg ml−1). After 3 min, the reaction was stopped by adding 100 μl ascorbic acid (25 mg ml−1) followed after 5 min by the addition of 50 μl human serum albumin (20%). The solution was then purified on a PD-10 column and eluted with 0.9% NaCl/ascorbic acid . The initial 2.7 ml eluate (2.2 ml sample volume and the first 0.5 ml) was discarded and the radiolabelled L19-SIP was collected in the next 3.0 ml.Preparation of 60 stationary phase and citrate buffer  mobile phase).Thin layer chromatography (TLC) was used to assess radiopharmaceutical purity  of radiolabelled antibody and dissected at 1, 3, 6 or 24 h after injection. For each time point, four mice were killed and the following organs were excised, weighed and counted in a γ-counter : blood, tumour, liver, spleen, kidney, intestine, heart, lung, brain, muscle, stomach and tail. The radioactive content of the different organs was expressed as the percentage of injected dose per gram of tissue (%ID g−1). Biodistribution experiments were analysed using Microsoft office Excel 2003.Biodistribution experiments with the 131I-L19-SIP were performed on SW1222 xenograft-bearing mice. To confirm that the 131I-labelled L19-SIP was selectively localizing to the tumour, and to compare localisation with 125I-labelled L19-SIP, a biodistribution study was first performed with the former. Each mouse was injected in the tail vein with 1.6 μg (0.75 MBq) of radiolabelled antibody, and groups of four mice killed at 3, 24 and 72 h after injection. Later time points  were included to investigate prolonged antibody retention, which is important for RIT efficacy. The following organs were excised, weighed and counted in a γ-counter: blood, tumour, liver, spleen, kidney, lung, colon, muscle, brain and stomach. The radioactive content of the different organs was expressed as %ID g−1.Therapy experiments with μg (55.5 MBq) 131I-L19-SIP per mouse. A second group of six mice were used as untreated controls.For the RIT experiments, a group of four mice was injected, intraperitonially, with 117.5 3 in volume. Therapy experiments were analysed using Microsoft office Excel 2003 and SPSS software. Survival was calculated using Kaplan–Meier analysis and showed significant correlation using the log-rank test (P=0.0034). Suppression of tumour growth was analysed using a non-parametric test (Mann–Whitney U-test). This showed a significant difference in tumour size between untreated and therapy groups at day 13 (P=0.038), which was sustained up until day 25 (P=0.011).During treatment, tumours were measured twice weekly for a period of approximately 140 days, and tumour volume was estimated as the measurement of length × width × height/2 , L19-SIP and perfusion (Hoechst 33342) over whole tumour sections, demonstrated that the antibody was associated with blood vessels in both peripheral and central regions of the tumour in the LS174T xenograft model . However125I and 131I. Preliminary studies showed that the protein could be labelled using the methods described above and a plateau of labelling was found at 4 min. Antigen binding of the labelled protein was found to be 91.7% for 125I-L19-SIP and 83.7% for 131I-L19-SIP. In addition, stability of the 125I-L19-SIP was assessed by TLC, and was found to be 99.2% after 5 days post-labelling.As mentioned in the Materials and Methods section, L19-SIP antibody was labelled following the IODO-GEN method with both 125I-labelled antibody (−1 reached a maximum of 7.9 (±1.3) at 6 h after injection. In comparison, this reached a maximum of 6.6 (±4) at 3 h in the LS174T tumours. At 24 h post-injection, the %ID g−1 was 4.5 (±1.2) and 5 (±0.6) for SW1222 and LS174T, respectively.To compare localisation of the L19-SIP antibody in the two colorectal xenograft models, biodistribution studies were carried out using antibody  and 3. IBlood stability studies showed that at 1 h 86.45±1.39% of activity was associated with active protein (binding to antigen), reducing to 62.01±4.9% after 24 h, in LS174T-bearing mice . This wa−1 h−1vs 122.6 for the LS174T tumours.Tumour to blood ratios for the two tumour models are shown in 131I-labelled L19-SIP was, therefore, carried out at 3, 24 and 72 h post-injection, to confirm the tumour specificity seen with 125I-labelled L19-SIP and to investigate long-term retention of antibody for RIT. Antibody distribution in tumour, blood and normal tissues is shown in −1, respectively. By 72 h, 1.7 (±0.8) %ID g−1 was still retained in the tumour.On the basis of these results, the SW1222 colorectal model was selected for a RIT study. A biodistribution study with the In addition, there was an increase in tumour to blood ratio of antibody over time, rising from 0.58 at 3 h to a maximum of 28 at 24 h. Although this ratio decreased by 72 h, it remained relatively high at 9.3 .131I-L19-SIP produced a significant effect on tumour growth and survival in SW1222 xenograft-bearing mice. Treatment with a single dose of 55.5 MBq mouse−1 inhibited subsequent tumour growth for a mean of approximately 14 days  had clearly localised to their targets by 1 h after injection. Interestingly, we have observed blood vessel fenestrations in the SW1222 subcutaneous tumours using transmission electron microscopy (data not shown), which may play a role in the extravasation of the L19-SIP antibody and binding to its target, although the involvement of transcytosis cannot be ruled out. These findings, therefore, indicate few barriers to efficient localisation of the L19-SIP antibody in our tumour model; an essential point to be considered when planning timing of future combined therapy regimes.When targeting antibodies to solid tumours, factors such as impaired microcirculation in newly formed vessels should be considered, as this may prevent antibody from rapidly reaching its target. Furthermore, the process of crossing the endothelial layer, which precedes the L19 antibody binding to the ED-B antigen on the abluminal surface of the vessel, could be retarded because of interstitial pressure. In addition, the endothelium itself has long been recognised as a natural barrier that may limit the uptake of antibodies in solid tumours. However, in the current study, we did not encounter these problems in our tumour models as both the 125I-L19-SIP antibody in the SW1222 xenografts in comparison to LS174T tumours  at 24 h post-injection (125I-L19-SIP (131I-L19-SIP (8.6 %ID g−1) in FaDu head and neck tumours at 24 h. Although higher radiolabelled antibody levels were found in blood at 3 h in the SW1222 tumour model, this fell dramatically by 24 and 72 h post-injection, indicating good blood clearance and produced high tumour to normal tissue ratios. Indeed, at 24 h, tumour to blood ratio was 28 : 1 . However, as the group size of the treated mice was small, this finding must be considered as preliminary and would need to be further confirmed in a larger group of mice.131I-L19-SIP.We have shown previously that the blood vessels distribution in SW1222 tumours compose approximately 8% of total tumour mass , and may be related to the high vascularity of the SW1222 tumours.No significant weight loss was observed in the treated mice indicating that the relatively high doses of radiation used were not toxic. Interestingly, the rapid blood clearance leading to high tumour to blood ratios obtained at the later time points (24 and 72 h) with 131I-L19-SIP, for example, repeated injection of the antibody. However, as the L19-SIP is targeting the tumour vessels, which compose only a small percentage of the tumour mass, it is unlikely that the therapy will be totally effective as a single agent. Future experiments will, therefore, concentrate on selecting the optimal combined complementary therapies to overcome the problems of tumour heterogeneity and treat the entire tumour. These combined therapies are most effective when targeting different regions of the tumour. For example, it could be combined with other forms of RIT that target the tumour cells rather than the vasculature (131I-L19-SIP with Cetuximab, an anti-EGFR antibody, enhanced the efficacy of the RIT without causing an increase in toxicity.In contrast to RIT using antibodies against tumour cell-associated antigens which are expressed only in certain cancers, such as carcinoembryonic antigen in colorectal tumours (125I-L19-SIP and Cy3-L19-SIP showed good tumour selectivity in two human colorectal xenografts with highly different morphologies, and that RIT with 131I-L19-SIP caused a significant tumour growth delay and improved survival. This in turn, provides the basis for future combined therapies with RIT.In conclusion, this study demonstrates for the first time that


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The vascularisation and perfusion of seven subcutaneously xenografted human glioma lines established from surgical specimens has been analysed using an anti-collagen type IV antibody to visualise the vascular walls in combination with a perfusion marker (Hoechst 33342). A computer-based digital image processing system was employed for quantitative analysis of the parameters. The vascular architecture of individual tumours belonging to the same tumour line showed a consistent similarity, while substantial differences occurred between the various tumour lines derived from different patients. Despite the presence of a large inter-tumour variation in vascular area as a proportion of the tumour area, this vascular parameter clearly showed tumour line-specific characteristics. The perfused fraction of the tumour vessels also showed a large inter-tumour variation for all tumour lines ranging from 20% to 85%, but the majority of tumours of all lines had perfusion fractions of more than 55%. Despite large variation, the perfused vascular area as a proportion of the tumour cross-sectional area exhibited clear tumour line-specific tendencies. These observations suggest that consistent differences in vascular parameters are present between glioma xenograft lines, although the tumour lines all originated from histologically similar human high-grade gliomas. These differences may have important consequences for treatment and clinical behaviour of this type of tumour.


This study investigated the modulation of type I collagen gene expression in normal fibroblasts by breast tumour cells. Northern analysis of total RNA extracted from stages I, II and III breast tumour tissue revealed that collagen mRNA levels were elevated in stage I tumours compared to the adjacent normal breast tissues, whereas they were decreased in stages II and III breast tumours. This aberrant collagen gene expression was confirmed by non-radioactive RNA:RNA in situ hybridization analysis of 30 breast carcinomas which localized the production of type I collagen mRNA to the stromal fibroblasts within the vicinity of the tumour cells. In order to determine whether the tumour cells were directly responsible for this altered collagen production by the adjacent fibroblasts, breast tumour cell lines were co-cultured with normal fibroblasts for in vitro assessment of collagen and steady-state collagen RNA levels. Co-culture of tumour cells and normal fibroblasts in the same dish resulted in down-regulation of collagen mRNA and protein. Treatment of the fibroblasts with tumour-cell conditioned medium also resulted in decreased collagen protein levels but the mRNA levels, however, remained unaltered. These results suggested that the tumour cells either secrete a labile ‘factor’, or express a cell surface protein requiring direct contact with the fibroblasts, resulting in down-regulation of collagen gene expression. Modulation of the ECM is a common characteristic of invading tumour cells and usually involves increased production of collagenases by the tumour cells or stromal fibroblasts. This study showed that tumour cells were also able to modulate collagen mRNA production by stromal fibroblasts, which may facilitate tumour cell invasion and metastasis. © 1999 Cancer Research Campaign