P<0.01). Number of peritoneal metastases (P<0.05), tumour volume, (P<0.05), vessel counts (P<0.01), and tumour cell proliferation (P<0.01) were significantly reduced in angiopoietin-1-expressing tumours. Conditioned medium from angiopoietin-1-transfected cells decreased vascular permeability more than did conditioned medium from control cells (P<0.05). Our results suggest that angiopoietin-1 is an important mediator of angiogenesis and vascular permeability and thus could theoretically serve as an anti-neoplastic agent for patients with carcinomatosis from colorectal cancer.Angiopoietin-1 is an important regulator of endothelial cell survival. Angiopoietin-1 also reduces vascular permeability mediated by vascular endothelial growth factor. The effects of angiopoietin-1 on tumour growth and angiogenesis are controversial. We hypothesised that angiopoietin-1 would decrease tumour growth and ascites formation in peritoneal carcinomatosis. Human colon cancer cells (KM12L4) were transfected with vector (pcDNA) alone (control) or vector containing angiopoietin-1 and injected into the peritoneal cavities of mice. After 30 days, the following parameters were measured: number of peritoneal nodules, ascites volume, and diameter of the largest tumour. Effects of angiopoietin-1 on vascular permeability were investigated using an intradermal Miles assay with conditioned media from transfected cells. Seven of the nine mice in the pcDNA group developed ascites (1.3±0.5 ml (mean±s.e.m.)), whereas no ascites was detectable in the angiopoietin-1 group (0 out of 10)  87, 1182–1187. doi:10.1038/sj.bjc.6600598www.bjcancer.comCancer Research UK© 2002  Angiogenesis has been associated with aggressive disease in human colorectal cancer . AlthougThe effects of the angiopoietins on angiogenesis, tumour growth and vascular permeability remain controversial , two-fold vitamin solution , 1 mM sodium pyruvate, 2 mM L-glutamine and non-essential amino acids and incubated in 5% CO2–95% air at 37°C as previously described (The highly metastatic human colon cancer cell line KM12L4 was obtained from IJ Fidler (The University of Texas M.D. Anderson Cancer Center) . The construct was sub-cloned into a pcDNA3 vector  containing a hygromycin resistance gene , trypsinised  and re-suspended in 10% foetal bovine serum–modified Eagle's medium. Cells were counted and cell viability was assessed using the Trypan blue dye exclusion assay (>90% viability). Cells were resuspended in Hank's balanced salt solution for tumour cell inoculation into mice.For ad libitum throughout the experiment. Experiments were approved by the Animal Care and Use Committee at M. D. Anderson Cancer Center and met all the standards required by the UKCCCR guidelines for the welfare of animals in experimental neoplasia, as published  were acclimated for 1–2 weeks while caged in groups of five. Mice were housed as previously described (ublished .n=10 per group). The average body weight among groups was similar at assignment. Under sterile conditions, either Ang-1- or pcDNA (control)-transfected KM12L4 cells (1×106 cells) were injected into the peritoneal cavity of each mouse by means of a 30-gauge needle. An injection volume of 500 μl  was used to achieve a distribution adequate for peritoneal implantation of tumour cells. Animals were monitored daily (weighed once per week), and when mice in any group showed early signs of decreased mobility, all animals were killed by cervical dislocation after anaesthesia induction with pentobarbital . Body weight was determined, and ascites was collected by performing a lower midline incision and completely draining the intra-abdominal fluid into a flask. The ascites volume was then measured with a 1-ml tuberculin syringe. Incisions were then extended to allow photography of the peritoneal cavity and determination of the extent of peritoneal carcinomatosis. Peritoneal tumour nodules were counted, and the diameter of the largest tumour  was determined. Tumour tissue was then harvested, placed in either 10% formalin for paraffin embedding or snap-frozen in optimum cutting temperature solution  in preparation for subsequent immunohistochemical analyses.Mice were randomly assigned to one of two groups (n=3 per group) were injected intravenously with sterile 0.5% Evans blue dye (200 μl) via the tail vein. Ten minutes later, mice were given four separate intradermal injections at different sites into the dorsal skin – one each of CM-Ang-1, CM-pcDNA, PBS (negative control), and PBS plus VEGF (10 ng ml−1)  (positive control for increased vascular permeability). The injections were done using a 30-gauge needle, and the injection volume was 50 μl. Mice were sacrificed 20 min after intradermal injections by cervical dislocation following anaesthesia induction with nembutal. The dorsal skin of each mouse was harvested to permit visualisation of intradermal dye leakage. To determine the relative degree of vascular permeability, two dimensions (a and b) of the elliptically appearing area of dye leakage were obtained at each injection site and the area was calculated by the formula a×b×π. Densitometric analysis was performed using the NIH Image Analysis software (V1.62) from the National Institute of Health  as another means of quantifying the extent of dye leakage at the intradermal injection sites in each mouse. Digitally obtained images of the underside of the dorsal skin, including all injection sites, were converted to a gray-scale image, and dye density was analysed at each site .To investigate the effects of overexpression of Ang-1 by tumour cells on vascular permeability, the intradermal Miles assay was performed as described previously  and incubated for 20 min at room temperature in a protein-blocking solution consisting of PBS supplemented with 1% normal goat serum and 5% normal horse serum. The primary antibody directed against CD31 was diluted 1 : 800 in protein-blocking solution and applied to the sections, which were incubated overnight at 4°C. Sections were then rinsed in PBS and incubated for 10 min in protein-blocking solution before the addition of peroxidase-conjugated secondary antibody. The secondary antibody used for CD31 staining (peroxidase-conjugated goat anti-rat IgG) was diluted 1 : 200 in protein-blocking solution. After incubation with the secondary antibody for 1 h at room temperature, the samples were washed and incubated with stable diaminobenzidine substrate . Staining was monitored under a bright-field microscope, and the reaction was stopped by washing with distilled water. Sections were counterstained with Gill No. 3 haematoxylin solution  and mounted with Universal Mount (Research Genetics). CD31-stained vessels were counted at 50× magnification at four different quadrants of each tumour , and average vessel counts were calculated. For all immunohistochemical procedures, the primary antibody was omitted as a negative control.Antibodies for immunohistochemical analyses were obtained as follows: rat anti-mouse CD31 antibody from PharMingen  and peroxidase-conjugated goat anti-rat IgG from Jackson Research Laboratories . Tumours that had been frozen in optimum cutting temperature solution were sectioned at 8-μm intervals, mounted on positively charged slides and air-dried for 30 min. Tissue sections were then fixed in cold acetone, followed by 1 : 1 acetone/chloroform and acetone, and then washed with PBS. Specimens were then incubated with 3% H2O (v v−1)), rehydrated in PBS (pH 7.5) and microwaved for 5 min for antigen retrieval. Tumour sections were stained for proliferating cell nuclear antigen (PCNA) using mouse anti-PCNA clone PC10 DAKO A/S . Immunohistochemical procedures were performed as described previously  and allowed to dry overnight at room temperature. Sections were deparaffinised in xylene, treated with a graded series of alcohol washes . Fisher's exact test was applied for comparing incidences of peritoneal carcinomatosis and ascites formation.All statistical analyses were done using InStat Statistical Software version 2.03 , with P=0.07).To study the effect of Ang-1 on peritoneal implantation and growth of human colon cancer cells, pcDNA- and Ang-1-transfected KM12L4 cells were injected into the peritoneal cavities of nude mice. Thirty days after tumour cell inoculation, three of the control (pcDNA) mice showed decreased mobility (secondary to ascites), and the experiment was terminated. . Peritoneal carcinomatosis developed in seven (78%) of the nine mice in the control group, compared to three (30%) of the 10 mice of the Ang-1 group. However, the incidences of development of carcinomatosis were not significantly different (P<0.01) . Large tumours (6–15 mm) were detectable at frequency of 13.4±3.3 nodules/mouse in the pcDNA group and compared to 0.5±0.3 nodules/mouse in the Ang-1 group (0.5±0.3) (P<0.05). As tumour growth beyond 1–2 mm is angiogenesis dependent, measurement of the largest tumour was a consistent and unbiased reflection of the effects of Ang-1 on tumour angiogenesis. The maximal tumour volume was significantly less in the Ang-1 group than in the pcDNA group (P<0.05)  were calculated for each mouse. The total number of peritoneal metastases was significantly lower in the Ang-1 group than in the control group ((P<0.05) .P<0.01, Fisher's exact test). The ascites volumes in peritoneal metastases–bearing mice in the pcDNA group ranged from 0.3 ml to 5 ml, with a median ascites volume of 1.3 ml . The median ascites volume was significantly higher in the pcDNA group (P<0.01) than in the Ang-1 group as none of the Ang-1 mice developed ascites even though three of them had peritoneal tumours  than in mice injected with Ang-1-overexpressing KM12L4 cells (0 out of 10) (P<0.01)   ((P<0.01)  and 4.in vivo permeability assay to determine the direct effect of Ang-1 on vascular permeability. In this assay, effects on vascular permeability were investigated using conditioned media from Ang-1- or pcDNA-transfected KM12L4 cells. Injections with PBS and PBS plus VEGF served as a negative and positive control, respectively. Areas of intradermal dye leakage (a measure of vascular permeability) were significantly smaller at CM-Ang-1 injection sites than at CM-pcDNA injection sites  (P<0.05) ) was significantly lower compared to CM-pcDNA sites (76.8±2.9 pixels2×103) .To demonstrate whether the observed inhibition of ascites formation was attributable to reduced vascular permeability mediated by Ang-1 and not solely dependent on tumour burden, we used the Miles In the present study, overexpression of Ang-1 by human colon cancer cells inhibited tumour angiogenesis, growth of peritoneal metastases and ascites formation in an experimental model of peritoneal carcinomatosis. In studies using the HT29 human colon cancer cell line transfected with Ang-1, similar growth-inhibitory results were obtained (data not shown).The effect of Ang-1 on tumour neovascularisation and growth is controversial. The concept that the angiopoietins are important mediators of tumour growth was elegantly demonstrated by Holash and associates, who demonstrated the importance of the coordinated induction of the angiopoietins and VEGF in tumour angiogenesis . In this model, Ang-1 overexpression did not prevent peritoneal implantation of tumours, although peritoneal nodules were fewer and smaller in the Ang-1 group. Therefore, Ang-1 prevented angiogenesis dependent tumour outgrowth by inhibiting neovascularisation, which was reflected by measurement of the largest tumour in mice.As demonstrated in our permeability assay, Ang-1 levels in CM from Ang-1-transfected cells abrogated the increase of plasma leakage (dye leakage) of dermal microvasculature caused by tumour cell–derived growth factors (KM12L4 cells express relatively high amounts of VEGF (In conclusion, our study indicates that Ang-1 was an important mediator of tumour angiogenesis and vascular permeability in an experimental model of peritoneal carcinomatosis from human colon cancer. The inhibition of vascular permeability by Ang-1 is especially relevant in a tumour system associated with a great deal of morbidity due to ascites formation that accompanies carcinomatosis. Our studies suggest that Ang-1 could potentially serve as an anti-angiogenic/anti-permeability agent in the treatment of carcinomatosis from colorectal cancer.


Several years of experience have now accumulated in the targeting of anti-cancer agents so that we can take stock, identify problems and look for ways round them. Three major obstacles seem to limit present approaches. These are heterogeneity in the distribution of target molecules within the cancer cell population, the pharmacokinetic characteristics of macromolecules and host antibody response to foreign protein. An approach which we have been investigating uses antibodies or other vectors to carry enzymes which have no close human homologue to tumour sites. After clearing residual enzyme activity from the blood by one of several possible techniques, a relatively non-toxic prodrug is given. This prodrug is a substrate for the tumour located enzyme which results in the generation of a highly toxic molecule able to penetrate the tumour mass and cross cell membranes. Genetic engineering methods now offer the prospect of human immunoglobulins with tumour binding and catalytic sites having the potential to minimise host response. Whether this can be achieved depends on having antibodies with adequate specificity and our ability to develop enzyme-prodrug systems with the required characteristics. Early results encourage us to think progress can be made in this direction.


Intracardiac injection, in hooded Lister rats, of syngeneic MC28 sarcoma cells never induced tumour growth in normal bowel. Tumour growth occurred at the site of a colonic anastomosis if surgery preceded tumour injection but not if it followed tumour injection, even by as little as 1 h. Maximum enhancement of tumour growth occurred when the healing process had progressed between 2 and 8 days, with a peak at 5 to 7 days. The enhancing effect was largely over by the time the healing had progressed 14 days. The syngeneic OES5 breast carcinoma also grew at colonic anastomoses when surgery preceded tumour injection by 5 days, but not in normal colon. The MC28 sarcoma also grew at ileal anastomoses but not in the normal ileum after intracardiac injection. By injecting radiolabelled sarcoma cells, an estimate of the probability of a single bloodborne tumour cell lodging at a colonic anastomosis and leading to a tumour deposit was calculated to be of the order of 1:43 whereas the probability of the cell lodging in normal colon and causing a deposit is less than 1:4 x 10(4).


The immunotherapeutic value of tumour extracts or B.C.G. in preventing either the occurrence of primary tumours or the recurrence of tumours in surgically resected animals has been examined. A transplantable methylcholanthrene induced tumour in DBA/2J mice was used. Neither tumour extract nor chemically modified extract was effective in preventing tumour growth in immunized animals, even though the mice demonstrated measurable levels of cell mediated tumour immunity at the time of tumour challenge. The frequency of tumour recurrence after resection of small tumours (about 1·0 g) was significantly lowered by treatment of the mice with a combination of B.C.G. and either modified or unmodified tumour extract. The frequency of recurrence after resection of large tumours (about 2·5 g) was not affected by any form of immunotherapy although the survival time of treated animals was significantly prolonged. The immunological status of animals with small and large tumours was examined and it was shown that mice with 1·0 g tumours have unimpaired mitogen responsiveness and measurable tumour specific immunity, whereas mice bearing large tumours (2·5 g) have a markedly impaired immune system.


The impact of TP transfection on tumour growth was relatively modest and concerned only the initial stages of tumour expansion. Staining of TP tumours for endothelial (factor VIII) cells was always higher than controls. The staining ratio (TP/controls) tended to be reduced as tumours increased in size. The stability of TP expression was checked both in vitro (TP activity measurement) and in vivo (RT-PCR determinations) and there was no loss of TP expression over time which could be advanced to explain the progressive weakening of the impact of TP overexpression on both tumour growth and neoangiogenesis. © 2001 Cancer Research Campaign http://www.bjcancer.comTumoral thymidine phosphorylase (TP) appears to play a dual role by being involved in neoangiogenesis and by activating 5FU prodrugs at the tumoral target site. The aim of the study was to investigate more thoroughly these potential physiological and pharmacological roles of TP. A rat carcinoma cell line (PROb) was transfected with TP/PD-ECGF in order to study the effect of the overexpression of this enzyme (1) on the sensitivity of cells to 5′DFUR and 5FU in vitro and (2) on tumour growth in vivo by using a syngenic tumour model in the BDIX rat . Cytotoxic effects of 5′DFUR, and to a lesser extent those of 5FU, were enhanced in TP clones as compared to control cells: there was a highly significant correlation between TP activity and in vitro sensitivity to 5’DFUR (r


Co-administration of both the antibodies with radiation increased the radiosensitivity of U87MG, resulting in a significant delay of subcutaneous (s.c.) tumour growth. Furthermore, the addition of antibodies to the radiation decreased brain tumour sizes and is inhibited by 40–80% the increased tumour cell invasion provoked by radiotherapy, although promoted tumour cell apoptosis. Whereas nimotuzumab led to a reduction in the size of tumour blood vessels and proliferating cells in s.c. tumours, cetuximab had no significant antiangiogenic nor antiproliferative activity. In contrast, cetuximab induced a more marked inhibition of EGFR downstream signalling compared with nimotuzumab. Moreover, both antibodies reduced the total number of radioresistant CD133+ cancer stem cells (CSCs). These results were encouraging, and showed the superiority of combined treatment of mAbs to EGFR and radiation over each single therapy against glioblastoma multiforme (GBM), confirming the role of these drugs as radiosensitisers in human GBM. In addition, we first showed the ability of mAb specifics against EGFR to target radioresistant glioma CSC, supporting the potential use in patients.As epidermal growth factor receptor (EGFR) has been reported to be a radiation response modulator, HER inhibitors are regarded to act as potential radiosensitisers. Our study examined the role of nimotuzumab and cetuximab both, the two monoclonal antibodies (mAbs) to EGFR, as radiosensitisers in a murine glioma model  Glioblastoma multiforme (GBM) remains a deadly disease with a median survival of <1 year despite all therapeutic efforts based on the aggressive conventional anticancer therapies  .Among EGFR inhibitors, nimotuzumab (TheraCIM h-R3) and cetuximab (IMC-C225) have undergone an extensive evaluation in different tumour locations, showing activity when administered with ionising radiation  is a human GBM cell line. Cells were grown in a 1+1 mixture of Eagle's minimum essential medium and Basal medium,  containing 2 mThe human–mouse chimeric anti-EGFR monoclonal antibody (mAb) cetuximab was provided by ImClone Systems, Inc. . The humanised anti-EGFR mAb nimotuzumab was generated at the Centre of Molecular Immunology  as primary antibody, which is specific for the Ki-67 protein, a marker of proliferating cells, followed by incubation with streptavidine/HRP (DakoCytomation). At least 5 high-power fields were counted and scored per sample. To score a tumour cell as positive for Ki-67, a nuclear staining was required. For this methodology, tumour slices were fixed with Fix I solution , 1 M HEPES, 25% glutaraldehyde, 1% glucose). All other immunohistochemical stains were performed on paraformaldehyde-fixed, cryostat 5-μm tissue sections using standard techniques. The following primary antibodies were used: anti-human CD133/1 (1 : 10 dilution), anti-human EGFR (1 : 500 dilution) and anti CD31/PECAM-1 (1 : 100 dilution). After washing, corresponding HRP-conjugated secondary antibody were used to detect antibody–antigen reactions. The negative controls consisted of duplicate sections of the same specimens, in which the primary antibody had been excluded and replaced with PBS or negative control immunoglobulin. The sections were visualised with 3,3′-diaminobenzidine as a chromogen and counterstained with Mayer's hematoxylin. Representative tumour sections were identified on a light microscope  with an ocular magnification of × 40 evaluating 4–5 tumours from each group for the corresponding analysis. To score a tumour cell as positive, a complete membrane staining was required for EGFR and CD133. Total area of the endothelial blood microvessels was calculated using the software (Axio Vison 4.5). Fluorescent terminal-deoxynucleotidyl-transferase-mediated nick end labelling (TUNEL) to evaluate apoptosis was done as per the manufacturer's instructions . Slides were analysed in a drop of PBS with a fluorescence microscope  in five random fields at × 40 magnification.Until analysis all the specimens were shock frozen and stored in nitrogen. Immunostaining was performed using 5-Western blotting experiments were performed as described (nu/nu) were obtained from Charles River . The mice were housed and maintained under aseptic conditions in facilities approved by the German Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the German Animal Protection Law, and their use was approved by the local responsible authorities. Animals met the requirements of the 7 U87MG tumour cells subcutaneous (s.c.) into left flank and 2 × 104 cells intracranially into the right hemisphere of mouse brains with the help of a stereotactic device. Tumour volume from flank's were determined from direct measurement with callipers and calculated according to the formula as follows: 0.5 × (large diameter) × 2. Relative tumour volumes (RTV) were calculated by referring the median volumes of each day to the first measurement (set to 1) as described (−1) mice in the control and radiation-alone groups were injected with saline solution. For radiation groups, animals were exposed to a total dose of 3.0 Gy of total body radiation fractioned in 1.0 Gy weekly. In the combination group the antibodies were administered 6 h before radiation therapy. All animals were killed by day 28 when tumour weight from the control group exceeded the 10% of total animal weight. These studies were repeated twice both for subcutaneous and intracranial tumours.Female athymic mice  were prepared and stained with cresyl violet. At the area of largest dimensions tumour diameters and perimeters were determined by computer-assisted image with the help of a microscope (Zeiss Axioskop 40). Subcutaneous tumours were snapping frozen and stored at −80°C for additional analyses.The size of intracranial tumours was determined. For that purpose, brains were collected and shock frozen in 2-methylbutane. Sequential cryosections . The significance of differences between groups was compared using a Kruskal–Wallis test. The significance of differences in groups was compared using Dunn′s multiple comparison test. Differences were considered significant if Human U87MG GBM cell line sensitivity to radiation is influenced by the experimental conditions (vs control (3.5). In contrast, radiation and antibodies each as a single modality treatment did not affect tumour growth profiles compared with controls. Further, no significant decreases in body weight were observed in mice in any of the six treatment groups. These results suggest that nimotuzumab and cetuximab may enhance the antitumour activity of radiation in U87MG tumours in vivo.Here, we explored the combinatorial effect of the EGFR targeted therapy based on mAbs with radiotherapy. U87MG cell line was xenografted both in the flanks and in the brains of NMRI nude mice. All animals injected with the tumour cells developed tumours. The mice were then treated with either nimotuzumab or cetuximab, radiation or both modalities during 3 weeks. P>0.05), as well as each antibody given alone. In contrast, only the combination of each antibody with radiation reduced significantly (P<0.05) the growth of orthotopic tumours; for example: tumour perimeter (mean±s.e.m.) for h-R3+RT (5.2±0.9) and C255+RT (5.4±1.2) versus control (11.1±2.1). More interesting, a histopathological analysis of intracranial tumour sections showed a strikingly more invasive growth pattern in mice treated with radiotherapy alone compared with antibodies-based therapies. These tumours were usually surrounded by numerous small-satellite tumours , among different group of treatments  in the control group was negligible, sharply contrasting with the different groups of treatment; however, no statistical differences between them were found .P<0.05) on cell proliferation was reached between the two groups of combination.We next examined whether both antibodies might enhance the antiproliferative effect of radiation by analysing the Ki-67 expression. The analysis of the proliferative activity in these tumours showed that, nimotuzumab enhanced the inhibition of proliferation caused by radiation by 56.5% in the combination group, compared with the antibody alone or radiotherapy  vascular niche, representing a potential target for treatments of brain tumours  (in vivo (The antitumour effect of anti-EGFR mAbs in combination with radiotherapy has been thought to result from the enhancement of the inhibition of EGFR signalling, increasing the cytotoxic effect of the radiation (Interestingly, CSC has been identified as a new cellular subpopulation of brain tumour cells with potent tumourigenic activity (A question emerging from this study was the lack of therapeutic response when nimotuzumab was given alone, despite shown a significant antiangiogenic activity. The reason is not fully understood, but it is likely that a combination of different mechanisms is needed to translate into a relevant therapeutic response. Indeed, the combined treatment with nimotuzumab and radiotherapy enhanced the antiproliferative activity of both drugs compared with individual therapies. Similarly, the absence of activity of cetuximab given alone, despite the abrogation of the EGFR signalling, suggest that it is at least in part responsible for the enhancement of the cytotoxic effects of radiation, but not enough to produce a substantial therapeutic response.More important, both antibodies displayed capacity to inhibit the tumour cell invasion provoked by the radiotherapy. It has established that the blockade of angiogenesis leads to compensatory mechanisms by which tumours may recruit their own vascular supply from pre-existent vessels and grow despite the absence of neovascularisation, a phenomenon named as vessel co-option (β, circulating cancer cells and tumour metastases by a direct effect of the TGF-β on the cancer cells may also contribute to explain that unexpected biological effect of radiotherapy (Glioma invasiveness has been linked to the development of local tumour satellites modulated after therapeutic interventions . A curreβ, proteins of the BCL-2 family, integrins and metalloprotease activity may promote glioma invasion (β could mediate EGFR transactivation, and subsequently activate ERK and p38MAPK through PKC (Furthermore, the possible interrelations of TGF-β on promote tumour cell invasion and metastasis after a radiation therapy.In addition, radiation therapy may enhance the EGFR intracellular activation pathways after irradiation, which in turn may contribute to enhance the tumour invasiveness . EGFR inin vivo. This work may enhance our preclinical understanding of the interaction of radiation and the inhibition of the EGFR to increase radiation-induced tumour toxicity. This information further serves as preclinical background for clinical investigations involving anti-EGFR antibodies with radiation in human GBM, a tumour relatively resistant to the treatment with other well known anti-EGFR agents, such as Iressa and Tarceva (In summary, we have provided evidence that nimotuzumab and cetuximab, two specifics mAb to EGFR, may increase the radiosensitivity of human U87MG tumour, enhancing the antitumour efficacy of radiation


The effectivenss of maltose tetrapalmitate (MTP) as an antitumour immune adjuvant was verified by its comparison with other known immunopotentiators, namely BCG, Corynebacterium parvum, levamisole and pyran copolymer. Copenhagen x Fisher 344/CRBL F1 hybrid male rats inoculated s.c. with the Dunning R3327A prostatic adenocarcinoma were used as the test system. All animals treated with immunoadjuvants showed a delay in tumour appearance and inhibition of early tumour growth. MTP was found to be the most effective, followed by levamisole, BCG, pyran copolymer and C. parvum in order of decreasing efficacy. Intratumoral treatment of small or large s.c. tumours with BCG, MTP and C. parvum was ineffective in our cases. However, this treatment was effective with MTP and BCG if they were used against a differentiated form of R3327 tumour. MTP and levamisole were found to be equally effective when given orally in drinking water. Experiments involving surgical excision of tumours followed by MTP therapy in two s.c. implanted animal tumour models  showed beneficial effects of MTP on local tumour recurrence and tumour growth. Pre- and postoperative MTP treatment was at least as effective as postoperative MTP treatment alone.


Recently, Ewing's tumours have been shown to carry specific hybrid transcripts resulting from the fusion of the EWS gene with FLI-1 or ERG genes. Based on the sensitivity and specificity of the detection of these alterations by the reverse transcriptase-polymerase chain reaction technique, we have developed an assay to search for small numbers of Ewing cells in various sites from patients with Ewing's tumour. This method enables the detection of fewer than one tumour cell per million blood mononuclear cells. A total of 28 primary sites and 51 peripheral samples from 36 patients were investigated. Tumour cells could be detected in 4/18 blood samples, 4/15 bone marrow aspirates and 2/18 peripheral stem cell harvests. EWS/FLI-1 and EWS/ERG transcripts being observed in eight and two cases respectively. The type of fusion transcript detected in peripheral site(s) was identical to that observed in the primary site. At diagnosis 5/16 patients (31%) demonstrated either circulating tumour cells or/and occult bone marrow metastasis. After induction therapy, tumour cells were detected in 3/21 patients. This highly sensitive method should be a relevant tool to allow a more accurate clinical assessment of the dissemination of Ewing's tumours.


Ktrans was obtained, which reflects vascular permeability and perfusion. Mice were imaged immediately before, and following, acute treatment with ZD6474 . Whole tumours were analysed to obtain mean Ktrans values, and a histogram approach was used to examine intratumour heterogeneity. Reproducibility of Ktrans measurements gave inter- and intra-animal coefficients of variation of 40 and 18%, respectively. Dose-related reductions in Ktrans were evident following acute ZD6474 treatment. A Ktrans reduction of approximately 30% (P<0.001) was evident with 50 and 100 mg kg−1 ZD6474, a reduction of 12.5% (P<0.05) at 25 mg kg−1, and a reduction that did not reach statistical significance at 12.5 mg kg−1. A correlation between this dose response and the growth inhibitory effect of ZD6474 following chronic treatment was also observed. The histogram analysis of the data indicated that ZD6474-induced a Ktrans reduction in both the most enhancing rim and the core of PC-3 tumours. Dynamic contrast-enhanced magnetic resonance imaging may have a role in assessing the acute effects of VEGF signalling inhibition, in clinical dose-ranging studies.Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), using gadopentetate dimeglumine, was used to monitor acute effects on tumour vascular permeability following inhibition of vascular endothelial growth factor-A (VEGF-A) signal transduction. Mice bearing PC-3 human prostate adenocarcinoma xenografts were treated with ZD6474, a VEGF receptor-2 (KDR) tyrosine kinase inhibitor. The pharmacokinetic parameter  Vascular endothelial growth factor-A (VEGF) has a key role in inducing the pathological angiogenesis required to sustain solid tumour growth. Vascular endothelial growth factor-A expression is elevated in tumours in response to a variety of stimuli  being measured as an indicator of vascular permeability  and can be correlated with the effect on PC-3 tumour growth induced by more chronic ZD6474 treatment. This study also highlighted the utility of using a histogram method to analyse DCE-MRI data, with greater effects on Ktrans being evident in the core of the tumour.The data indicate that significant reductions in in vivo studies were performed in full compliance with the UK Animals (Scientific Procedures) 1986 Act. PC-3 human prostate adenocarcinoma xenografts were established in athymic mice as described previously  × √ (length × width) × (π/6), taking length to be the longest diameter across the tumour and width the corresponding perpendicular. We have previously shown that there is a good correlation between tumour volumes determined by caliper and MRI measurement  solution of polyoxyethylene sorbitan monoleate in deionised water and the animals were dosed once daily by oral gavage at 0.1 ml 10−1 g body weight. In tumour growth studies, ZD6474 or vehicle were administered once daily for 11 days.All VE). The transfer constant for this movement (Ktrans) is dependent on the blood flow, the surface area and the permeability of the vasculature. The concentration of Gd-DTPA in plasma and tissue is determined from the change in T1 relaxation and the relaxivity of Gd-DTPA in plasma and tissue. The input function measured in the abdominal aorta is assumed to be equivalent to that in the tumour vasculature.The Tofts and Kermode model considers contrast moving passively between two compartments; plasma and the extracellular space  for approximately 2 h during which they were mounted in a lidded plastic cradle fitted with a thermocouple and electrocardiogram electrodes. Under a flow of warm air, a heparinised 26-gauge catheter was inserted into the tail vein and attached to a syringe containing 30 mM Gd-DTPA  in sterile water. The mice were then transferred, in the cradle, to the bore of a horizontal magnet, where their temperature was maintained at 38.0±0.5°C by a flow of warm air, and their heart rates were monitored.Three separate experiments were carried out that included control groups given vehicle alone, and one or more treatment groups given ZD6474 at 12.5, 25, 50 or 100 mg kgUNITYInova, Varian Medical Systems, Palo Alto, CA, USA). The precontrast acquisition protocol comprised a heavily T2-weighted multislice fast spin-echo sequence with a repetition time (TR) of 3000 ms and an effective echo time (TE) of 120 ms. Four slices were obtained covering the whole tumour volume with a resolution of 0.625 × 0.312 × 2 mm (this resolution was employed throughout with a matrix of 512 × 256 × 4). This was followed by a saturation-recovery sequence for T1 mapping of the tumour that consisted of a series of 4 multislice spin-echo image sets . Repeated T1-weighted spin-echo imaging  was then used to generate multislice dynamic data. Spin-echo images were employed as opposed to the faster gradient-echo, as spin-echo approaches are less prone to confounds from radiofrequency inhomogeneity in assessment of T1 changes than are gradient echoes. Spin -echoes were also found to give a better signal-to-noise ratio and therefore increased the accuracy of voxel by voxel data fitting. Following acquisition of five dynamic data sets, Gd-DTPA solution (0.01 ml g−1 body weight) was manually injected over 3 s into the tail vein of the animals. The dynamic run then continued with repetitions every 16 s for 11 min after the injection, providing 40 image sets. A calibration curve was prepared by measuring the longitudinal relaxation time (T1) of filtered mouse blood plasma solutions containing different Gd-DTPA concentrations. The concentrations ranged from 0.0–1.8 mM and were measured at 38°C using a saturation-recovery spin-echo imaging sequence. The calibration curve was used to obtain the relaxivity of Gd-DTPA. A vascular input function, obtained previously in the same animal model, and modelled as a biexponential decay, was employed:Dynamic contrast-enhanced magnetic resonance imaging was carried out on an instrument incorporating a 63 mm bore quadrature birdcage coil and a 4.7 Tesla horizontal-bore magnet interfaced to a spectrometer −1=16.62 kg l−1 represents the plasma volume of the mouse.where T2-weighted images to include tumour but exclude surrounding skin. Time courses for contrast enhancement for each voxel within the whole tumour were used to produce parametric maps for analysis using the pharmacokinetic model of Ktrans), and of the volume of extravascular extracellular space per unit volume of tissue (VE) , running on a unix workstation. Region of interest comprised entire tumour volumes and were defined using the t-test) and by comparing mean individual animal data obtained pre- and post-treatment (paired t-test). The reproducibility of each pairwise comparison in control animals was assessed from the test-retest coefficient of variation (CV). For each subject, i, the CV is the standard deviation, σi, for the two measurements on that subject, divided by the mean volume, μi, for the subject. The overall test-retest CV for a group of N subjects is then:Tumour data were analysed both by grouping all values within treatment groups  and cores (bottom 80%). Changes in the number of fitted voxels over the 20% threshold pre- and post-treatment with ZD6474 were calculated on a mouse-by-mouse basis and examined using a paired t-test. This enabled the effect of drug treatment on the most rapidly enhancing rim  and the slower enhancing core  to be examined. The exercise was repeated to examine the ZD6474-induced changes in the number of voxels below the 20% threshold. A significance level of 0.05 was used throughout.To address the heterogeneous enhancement in tumours (i.e. a rapidly enhancing rim and slowly enhancing core), a novel histogram-based analysis was made by pooling all the An example of a baseline tumour image (single slice), preceding ZD6474 treatment, is shown in Figure 1A sample kinetic plot of contrast agent concentration for single voxels (selected at random) in the rim and the core of a tumour are shown in Figure 2T2-weighted fast spin- echo images and time courses for enhancement through the tumour enabled voxelwise calculation of pharmacokinetic parameters. There were no significant differences in repeat measurements of mean Ktrans (P=0.40) or VE (P=0.10) made on the same tumour. There was some variability between animals, with inter animal CVs of 40 and 18% for Ktrans and VE, respectively  that may reflect the extent of tumor necrosis. The Ktrans data for a single tumour and illustrates the drug-induced changes seen. Using the histogram analysis approach described in the Materials and Methods, significant reductions in Ktrans were seen in both the 20% most-enhancing (rim) and the remaining least-enhancing (core) parts of tumours  PC-3 tumours , with mean tumour volumes being 70 and 51% smaller, respectively, than their pretreatment volumes. In contrast, the predominant effect from treatment with 25 mg kg−1 ZD6474 was a reduction in the rate of tumour growth, with regressions (of 13 and 40%) only being evident in two out of seven tumours.Chronic once daily oral administration of ZD6474 for 11 days produced dose-related effects on the growth of well-established . A reproducibility study examining vascular permeability measurements in patients with solid tumors indicates that DCE-MRI assessment using Gd-DTPA may be even more reproducible in man (The DCE-MRI study described here used Gd-DTPA to measure the dynamic parameter −1 day−1 or less (3 volume), it is possible to induce regression with chronic ZD6474 treatment. This phenomenon is believed to result from the inhibition of VEGF-signalling alone , while chronic treatment induced tumour regressions, with equivalent effects being apparent after 7 days of dosing. However, 100 mg kg−1 ZD6474 did eventually have a greater effect on tumour volume, after 11 days of treatment. A smaller but significant effect on Ktrans (reduction of 12.5%) was apparent following acute treatment with 25 mg kg−1 ZD6474, but without a significant effect on VE. The predominant effect of chronically administering this dose of ZD6474 was to inhibit the rate of PC-3 tumour growth. In contrast, acute treatment with 12.5 mg kg−1 ZD6474 did not produce a statistically significant effect on Ktrans or VE. Collectively, these data suggest that in this tumour model, a relationship exists between the dose of ZD6474 administered, the MRI parameters Ktrans and VE (measured after acute treatment) and the effect on tumour growth (following chronic dosing).Dynamic contrast-enhanced magnetic resonance imaging enabled an effect on VE is unlikely to be used to monitor changes in vascular permeability, but is reported for interest. Since acute exposure to ZD6474 (50 and 100 mg kg−1) would not be expected to reduce the volume of the interstitial space significantly, the reductions in VE observed might be a consequence of significantly reduced vascular permeability, which could limit contrast agent availability to the extracellular space. In principle though, VE could increase or decrease in response to changes in transK, and VE is not well determined where Ktrans is low. Indeed, some high values for VE were observed in the tumour core where Ktrans was reduced.vs Ktrans can be obtained acutely with a VEGF-signalling inhibitor, using Gd-DTPA. Small molecular weight contrast agents have recently been used in two other preclinical studies to examine the effect on transK following inhibition of VEGF signalling. In the first of these, an antibody to VEGF was administered to athymic rats bearing small intracranial tumours  following chronic administration (21 days) in a murine syngeneic renal tumour model and a reduction in permeability of approximately 50% was reported (Ktrans and determine responses following acute treatment with VEGF-signalling inhibitors.This is the first preclinical study to indicate that dose–response information Ktrans maps are not well developed and generally manually selected ROI are used. Examination of the distribution of data using a histogram analysis, as used in this study, could provide additional information on intratumour heterogeneity, reduce sampling errors and improve understanding of the mechanisms behind tumour responses to treatment. The histogram threshold analysis indicated that, following ZD6474 treatment, significant reductions in Ktrans were evident in both areas of the tumour, but tumour cores were affected to a greater extent. Using manually selected ROI in xenografted human breast tumours, reduced vascular permeability has been seen to a similar extent in both the tumour rim and its centre, following administration of an anti-VEGF antibody (Analysis of the mean of all voxels within a tumour may not be optimal for assessing the effectiveness of anti-VEGF therapy using DCE-MRI, since highly vascular areas could respond differently from regions containing substantial necrosis. The tumour rim is known to be highly enhancing, and the centres of tumours are known to contain variable degrees of necrosis (Ktrans data can reveal additional spatial information of interest.In summary, this study suggests that DCE-MRI, using a small molecular weight contrast agent, could provide a method for examining permeability changes in response to acute treatment with an inhibitor of VEGF-signalling. The study also indicates that a histogram analysis of tumour


Primary human panceratic exocrine adenocarcinoma has been established in tissue culture and as xenografts in immune-deficient nu/nu mice. The cell line has a doubling time of 36 h and grows as a confluent monolayer together with a constant population of free-floating cells. Evidence of tumourigenicity was provided by growth on an early diploid fibroblast monolayer and in soft agar, and as solid tumours in immune-deficient nu/mu mice. Chromosome analysis of the cultured cells confirmed their tumour origin. Xenografts established from the cell line or directly from primary tumour tissue have retained a similar histology to the original tumour on serial transplantation. An electrophoretic study of exportable pancreatic digestive enzymes and a number of intracellular enzymes has shown that the cell line and xenografts maintain a human intracellular enzyme profile, but do not produce pancreatic digestive enzymes.


Metastasis, the spread and growth of tumours at secondary sites, is an extremely important clinical event, since a majority of cancer mortality is associated with the metastatic tumours, rather than the primary tumour. In spite of the importance of metastasis in the clinical setting, the actual process is extremely inefficient. Millions of tumour cells can be shed into the vasculature daily; yet, few secondary tumours are formed. The classical hypothesis explaining the inefficiency was a series of secondary events occurring in the tumour, resulting in a small subpopulation of cells capable of completing all of the steps required to successfully colonise a distant site. However, recent discoveries demonstrating the ability to predict metastatic propensity from gene expression profiles in bulk tumour tissue are not consistent with only a small subpopulation of cells in the primary tumour acquiring metastatic ability, suggesting that metastatic ability might be pre-programmed in tumours by the initiating oncogenic mutations. Data supporting both of these seemingly incompatible theories exist. Therefore, to reconcile the observed results, additional variables need to be added to the model of metastatic inefficiency. One possible variable that might explain the discrepancies is genetic background effects. Studies have demonstrated that the genetic background on which a tumour arises on can have significant affects on the ability of the tumour to metastasise and on gene expression profiles. Thus, the observations could be reconciled by combining the theories, with genetic background influencing both metastatic efficiency and predictive gene expression profiles, upon which, subsequently, metastasis-promoting mutational and epigenetic events occur. If the genetic background is an important determinant of metastatic efficiency, it would have significant implications for the clinical prediction and treatment of metastatic disease, as well as for the design of potential prevention strategies. Metastasis is an extraordinarily complex process. To successfully colonise a secondary site, a cancer cell must complete a sequential series of steps before it becomes a clinically detectable lesion. These steps include separation from the primary tumour, invasion through surrounding tissues and basement membranes, entry and survival in the circulation, lymphatics or peritoneal space, arresting in a distant target organ, usually, but not always .This process is of great importance to the clinical management of cancer, since the majority of cancer mortality is associated with metastatic disease rather than the primary tumour . In mostIn spite of the prevalence of secondary tumours in cancer patients, the metastatic process is an extremely inefficient process. To successfully colonise a distant site, a cancer cell must complete all of the steps of the cascade. Failure to complete any step results in the failure to colonise and proliferate. As a result, tumours can shed millions of cells into the bloodstream daily ; yet, veThe first suggestion of the role of genetic background as a critical determinant of metastatic potential was derived from transfection experiments. Introduction of proto-oncogenes can induce tumorigenicity and metastatic potential when transfected into NIH-3T3 cells. However, when the same oncogenes were transfected into cell lines derived from different strains of mice, metastatic potential, but not tumorigenicity, was lost 1 progeny were aged to permit tumour induction and potential metastatic dissemination. Subsequently, the lungs were examined to determine whether introduction of allelic variation had an affect on the density of pulmonary metastases, and a wide variation in metastatic efficiency was observed . These observations suggest that the propensity of a tumour to metastasise, and the predictive gene expression profile, is at least in part set by the combination of subtle changes in gene function, mediated by polymorphisms in coding sequence, splice sites, promoters, and enhancers, before tumour initiation. Subsequently progressive events such as translocations, deletions, etc., occur to produce rare cells that are capable of completing the metastatic process. The allelic background of the tumour would also likely influence what specific secondary events would be necessary in each individual host genotype to successfully complete the metastatic cascade.The truth is likely to be a blend of the models, with additional variables added in. One of these variables is likely the affect of genetic background as a determinant of metastasis. As previously mentioned, we demonstrated that the genetic background on which a cancer arises has a significant affect on the ability of mammary tumours to successfully colonise the lung. In addition, we and others (Importantly, the genetic efficiency determinant not only exerts its affects within the tumour cell itself, but also in the primary tumour stroma as well as the microenvironment at distant sites. Target organ microenvironment is known to play an important role in metastasis formation . Tumour The growing evidence suggesting that the majority of tumour cells are capable of extravasating (These observations, particularly the microarray data, have important implications for metastasis detection and management. If genetic background is a major influence on metastatic potential, as measured by predictive gene expression patterns in normal and tumour tissue, it suggests that, like cancer susceptibility, there may be individuals or families present in the human population that are more susceptible to disseminated disease. It may therefore be possible to identify these individuals before they develop neoplastic disease, so that they might be more aggressively treated with neo-adjuvant therapies immediately upon diagnosis of the primary tumour. Alternatively, since tumour dissemination often appears to be an early event, it is theoretically possible that a chemoprevention regime might be developed that would prevent tumour metastasis before the primary tumour was clinically apparent, enabling the bulk of human cancer to be cured by surgical resection.In conclusion, the identity of the genomic elements in the host background modifying metastatic efficiency is currently unknown. They clearly warrant further investigations, since the majority of the genetically defined regions are not associated with known metastasis-suppressor genes. The metastasis suppressors that are associated with our genetically defined regions do not have any apparent molecular defects nor expression level differences between the high and low metastatic genotypes (


The effect of Photofrin-based photodynamic therapy (PDT) and adjuvant treatment with serum vitamin D3-binding protein-derived macrophage-activating factor (DBPMAF) was examined using a mouse SCCVII tumour model (squamous cell carcinoma). The results show that DBPMAF can markedly enhance the curative effect of PDT. The most effective DBPMAF therapy consisted of a combination of intraperitoneal and peritumoral injections (50 and 0.5 ng kg-1 respectively) administered on days 0, 4, 8 and 12 after PDT. Used with a PDT treatment curative to 25% of the treated tumours, this DBPMAF regimen boosted the cures to 100%. The DBPMAF therapy alone showed no notable effect on the growth of SCCVII tumour. The PDT-induced immunosuppression, assessed by the evaluation of delayed-type contact hypersensitivity response in treated mice, was greatly reduced with the combined DBPMAF treatment. These observations suggest that the activation of macrophages in PDT-treated mice by adjuvant immunotherapy has a synergistic effect on tumour cures. As PDT not only reduces tumour burden but also induces inflammation, it is proposed that recruitment of the activated macrophages to the inflamed tumour lesions is the major factor for the complete eradication of tumours.


Angiogenesis is a crucial event in carcinogenesis and its onset has been associated with premalignant tumour stages. In order to elucidate the significance of angiogenesis in different stages of epithelial skin tumours, we analysed the vessel density in ten normal skin samples, 14 actinic keratosis (AK), 12 hypertrophic AKs, and in nine early- and 16 late-stage squamous cell carcinomas (SCCs). Mean vascular density was quantitated by counting the number of CD 31-immunostained blood vessels and by morphometric assessment of stained vessel area by computer-assisted image analysis. The results from both methods were well correlated. Mean vascular density was similar in normal dermis and in AK, and only slightly elevated in hypertrophic AKs and early SCC stages (tumour thickness < 2 mm). Only late-stage SCCs infiltrating the subcutis exhibited a significant increase in vascularization. Vessel density was independent of tumour localization, degree of proliferation and inflammatory cell infiltration. Furthermore, tumour vascularization was not correlated with the expression of vascular endothelial growth factor, a major angiogenic factor, as revealed by in situ hybridization and immunohistochemistry. The restriction of enhanced vascularization to increased tumour thickness may be a major reason for the rather low metastatic spread of cutaneous SCCs. © 2000 CancerResearch Campaign


Cellular transport and retention of [18F]FETA were determined in vitro under air vs nitrogen. Biodistribution and metabolism of the radiotracer were determined in mice bearing MCF-7, RIF-1, EMT6, HT1080/26.6, and HT1080/1-3C xenografts. Dynamic PET imaging was performed on a dedicated small animal scanner. [18F]FETA, with an octanol–water partition coefficient of 0.16±0.01, was selectively retained by RIF-1 cells under hypoxia compared to air (3.4- to 4.3-fold at 60–120 min). The radiotracer was stable in the plasma and distributed well to all the tissues studied. The 60-min tumour/muscle ratios positively correlated with the percentage of pO2 values <5 mmHg  and carbogen breathing decreased [18F]FETA-derived radioactivity levels (P=0.028). In contrast, nitroreductase activity did not influence accumulation. Tumours were sufficiently visualised by PET imaging within 30–60 min. Higher fractional retention of [18F]FETA in HT1080/1-3C vs HT1080/26.6 tumours determined by dynamic PET imaging (P=0.05) reflected higher percentage of pO2 values <1 mmHg (P=0.023), lower vessel density (P=0.026), and higher radiobiological hypoxic fraction (P=0.008) of the HT1080/1-3C tumours. In conclusion, [18F]FETA shows hypoxia-dependent tumour retention and is, thus, a promising PET marker that warrants clinical evaluation.Development of hypoxia-targeted therapies has stimulated the search for clinically applicable noninvasive markers of tumour hypoxia. Here, we describe the validation of [ Hypoxia occurs to a variable extent in most rodent and human solid tumours . It resupO2 leads to poor outcome after treatment (α (HIF-1α3), and carbonic anhydrase IX have also shown promise (18F-labelled 2-nitroimidazoles such as [18F]FMISO (18F]EF5 (18F]FETNIM (18F]FAZA (N-(2-([18F]fluoroethyl)-2-(2-nitroimidazol-1-yl)-acetamide ([18F]FETA) (60Cu]ATSM (99mTc]HL-91 (123I]IAZA (Studies with fine needle oxygen electrodes provided proof that low pO2 <10 mmHg (pO2 <1 mmHg (1/7 of ∼−380 to −390 mV) for selective reduction and binding in hypoxic tumour cells; (ii) lipophilicity that is high enough to enable diffusion across cellular membranes to the site of metabolism , an extremely promising hypoxia probe for imaging by PET. We demonstrate for the first time in mouse models of cancer that the retention of [18F]FETA-derived radioactivity correlates with pO2 and radiation sensitivity.Of the numerous possibilities in nuclear medicine, appropriately labelled 2-nitroimidazole probes are particularly attractive for imaging tumour hypoxia -ethyl]-phthalimide 18F reaction using H218O enriched water , before cooling. In total, 15 mg of compound (I) was dissolved in 500 μl acetonitrile. This was added to the reaction vessel, which was sealed at 100°C for 10 min before evaporation of the acetonitrile. A measure of 75 μl hydrazine hydrate was added to the reaction vessel. [18F]fluoroethylamine (IV) was distilled from the reaction vessel  into a vial containing 4 mg precursor (II) in 400 μl of ice cold acetonitrile. The vial was sealed and stirred for 30 min at room temperature. The resulting product was purified by high-performance liquid chromatography (HPLC) (Waters μ-Bondapak C18 column) and eluted with water . The radioactive product having the same retention time as fluoroetanidazole was collected  from the end of bombardment was approximately 3 h.Unless otherwise noted, all reagents, including anhydrous solvents, were obtained from Sigma-Aldrich . The synthesis of [e method . The pree method . [18F]fled water . A measu18F]FETA was determined by measuring the octanol–water partition coefficient. For this, 80–100 μCi (2.96–3.70 MBq) of [18F]FETA was dissolved in distilled H2O to a final volume of 0.5 ml and 0.5 ml of octan-1-ol  was added. The resulting mixture was processed in an orbital shaker  for 10 min at 300 rpm and subsequently centrifuged for 30 min . Aliquots (200 μl) of the resulting top layer (representing [18F]FETA dissolved in octanol) and bottom layer ([18F]FETA dissolved in H2O) were taken and the radioactivity in these samples was measured using a Cobra II Auto-Gamma counter . Six octanol–H2O mixtures were analysed and the complete experiment was repeated once. The octanol–water partition coefficient was calculated by dividing the octanol-containing radioactivity by the water-containing radioactivity.The lipophilicity of [18F]FETA uptake by cells was studied in vitro in RIF-1 tumour cells. Uptake was defined as the transport of radiotracer into cells and its specific binding to cellular macromolecules (and glutathione). The cells were cultured (5% CO2 incubator at 37°C) in T75 flasks in RPMI growth medium  supplemented with 10% foetal bovine serum (Sigma) and antibiotics. Exponentially growing cells were harvested, dissolved in phosphate-buffered saline (PBS) and placed in a custom-made hypoxic chamber  or with air as control, each flowing at 2 l min−1. After pregassing for 8 min, the cells were incubated with 80–100 μCi (2.96–3.7 MBq) of [18F]FETA. No correction for the total amount of stable etanidazole in the reaction mixture was made between experiments. Immediately after addition of the radioactivity, and at 15, 30, 45, 60, 90, and 120 min, the cells were harvested and centrifuged (5000 rpm for 5 min) to obtain a pellet. The pellet was washed twice with PBS (5 ml) and centrifuged as above. The cell number in the resulting pellet was determined by counting cells that excluded trypan blue. The total radioactivity added to each hypoxic chamber and the cell-bound radioactivity were measured on a Cobra II Auto-Gamma counter  to determine the degree of radioactivity binding. Percentage of bound radioactivity was calculated as (bound radioactivity in 106 cells × 100)/total radioactivity. These experiments were carried out in triplicate and repeated independently three times.The inhibitory effect of oxygen on FETA . Mice were killed by exsanguination via cardiac puncture 60 min after injection of the radiotracer. Aliquots of heparinised blood were rapidly centrifuged (2000 g for 5 min) to obtain plasma. The radioactivity contained in tumour, liver, kidney, spleen, lungs, heart, small intestines, brain, muscle, bone, blood, plasma, faeces, and urine was determined in a Cobra II Auto-Gamma counter and expressed as % ID g−1 (percentage of injected dose per gram of tissue). A minimum of six mice was used for each tumour model.Anaesthetised tumour-bearing mice were injected intravenously via the lateral tail vein with 100 2, 5% CO2), which is known to decrease tumour hypoxia . Untreated RIF-1 tumour-bearing mice served as controls (n=8). Mice were injected with [18F]FETA and killed at 60 min postinjection (p.i.), as described above. Successful modulation of tumour pO2 was verified by means of OxyLite probes as described below.Biodistribution studies were also carried out in mice following modulation of tumour hypoxia. In RIF-1 tumour-bearing mice, the degree of tumour hypoxia was modulated by administration of carbogen gas  were placed on thermal platforms to maintain their body temperature at ∼37°C. Dynamic PET scans were acquired over 60 min after intravenous (i.v.) injection of 40–100 μCi (1.48–3.7 MBq) of [18F]FETA via a tail vein cannula. The imaging and image-data processing protocol has been recently described in detail elsewhere (vs radioactivity curve(s) (TACs) from the ROIs were averaged for each tumour and normalised to the integral of the heart cavity TAC. Altogether, four HT1080/1-3C and three HT1080/26.6 tumour-bearing mice were scanned.In parallel with the 60-min biodistribution studies above, the time course of [18F]FETA metabolites by HPLC. For this, plasma samples (100 μl) were deproteinated by adding 2.0 ml ice-cold methanol and centrifuged . Liver, muscle, and tumour samples were cut into small pieces and homogenised in 2.0 ml of ice-cold methanol using an Ultra-Thurrax homogeniser  and the resultant homogenate was centrifuged . The supernatants from plasma and tissues were evaporated to dryness in a rotary evaporator (50°C) under vacuum, reconstituted in 1.5 ml of mobile phase (methanol/H2O (20/80% v v−1)), centrifuged and filtered (0.2 μm). Unlike plasma and tissue samples, urine and gall bladder samples were diluted with 1.5 ml of mobile phase and clarified by filtration (0.2 μm). Aliquots of each filtrate (1.0 ml) and of the injected dose solution diluted in 1.5 ml of mobile phase were analysed by HPLC. The samples were separated on a C18μBondapak column  that was eluted with the aforementioned mobile phase at a flow rate of 2.0 ml min−1. The radioactivity of the eluents was monitored. Peak areas were integrated and corrected for physical decay and background radioactivity.Plasma, urine, liver, gall bladder, muscle, and tumour samples obtained from RIF-1 tumour-bearing C3H/Hej mice were assessed for putative FETA uptake in vitro under normoxic and hypoxic conditions, pO2 values, and ex vivo [18F]FETA retention in RIF-1 tumours treated with carbogen vs untreated controls, as well as differences between vessel density, clonogenic survival, and fractional radiotracer retention obtained from dynamic PET scanning in 26.6 and 1-3C subclones of HT1080 tumours. Associations between [18F]FETA tumour retention, pO2 values, and nitroreductase activities in the established tumour models were tested for correlations by means of linear regression analysis. pO2 values obtained from the OxyLite measurements were expressed as median values for all investigated mouse tumours. Survival fractions from clonogenic assays were calculated relative to the corresponding plating efficiency and are given as mean±1 s.e.m. Survival curves were fitted by regression analysis and compared at the midpoints of the curves. Unless stated, data are expressed as mean±1 s.e.m. The P-values of ⩽0.05 were considered significant.Statistical analyses were performed using the software SPSS for Windows, version 10.0.7 . Differences in 18F]fluoride, [18F]FETA yields were typically 0.6–3.0 mCi (22–111 MBq), which represents a final (decay corrected to end of bombardment) radiochemical yield of approximately 10–20%. High-performance liquid chromatography analyses of the [18F]FETA dose solutions were performed 60 min after preparation of the radiotracer. [18F]FETA eluted at ∼9.5 min. The radiochemical purity was 94.6±1.5% (n=4). The lipophilicity of [18F]FETA expressed as octanol–water partition coefficient was determined to be 0.16±0.01 (n=12).Beginning with 30–50 mCi (1.11–1.85 GBq) [18F]FETA in air vs nitrogen in RIF-1 cells. The uptake of the radiotracer by normoxic cells was very low. In contrast, there was a rapid time-dependent linear increase of [18F]FETA uptake by RIF-1 cells under nitrogen, which was 3.0- and 4.3-fold as compared to the normoxia values at 60 and 120 min p.i., respectively. In this model system the hypoxia-dependent increase in [18F]FETA uptake reached significance after 60 min  in lung, heart, brain, and bone; intermediate (5–8% ID g−1) in plasma, liver, spleen, small intestines, and muscle; and high (>8% ID g−1) in kidney, bile, and urine  eluted at a retention time of ∼9.5 min. Parent [18F]FETA comprised 88% of the total radioactivity in plasma at 10 min p.i. In tumour, a minor radioactive peak at a retention time of ∼6 min that comprised 18% of the total radioactivity was detected at 10 min p.i., whereas most of the radioactivity (92%) in the liver was associated with the metabolite(s) eluting at ∼6 min. At 60 min p.i., metabolite peaks with retention times of ∼6 to 7 min were detected in the plasma , muscle (50%), liver (99%), bile (82%), tumour (52%), and urine (26%)  and 60 min  after the administration of FETA retention, expressed as tumour-to-muscle ratio, was positively correlated with the relative frequency of pO2 values <5 mmHg. This correlation was found to be linear  and significant . In addition, tumour/muscle ratios obtained from the [18F]FETA biodistribution studies were significantly higher for the 1-3C subclone as compared to the 26.6 subclone .For all the tumour models, the relative FETA tumour retention or tumour pO2 obtained from OyxLite measurements.The activities of cytochrome 18F]FETA tumour retention to 4.70±0.26% ID g−1, compared to untreated controls . In tissues other than tumours, [18F]FETA retention was not altered by carbogen breathing. Administration of carbogen decreased the relative frequency of pO2 values <2.5 mmHg in comparison to untreated controls (40±11 vs 51±13%). However, these differences did not reach significance level (P=0.551).The ability to detect acute changes in tumour oxygenation was investigated in RIF-1 tumours after application of a physiological modulator, for which the resultant effect is well documented. Carbogen breathing decreased FETA is a suitable radiotracer for PET imaging of tumour hypoxia.The partial pressure of oxygen has been directly determined in accessible tumours such as cervix tumours, head and neck tumours, and sarcomas using Eppendorf oxygen electrodes (18F-labelled 1-(2-nitro-imidazolyl)-3-fluoro-2-propanol ([18F]fluoromisonidazole ([18F]FMISO) , consistent with the presence of a 2-nitroimidazole moiety  around −380 to −390 mV (F]FMISO) ); and (ie (0.046 ), but lo −390 mV . The lar18F]FETA accumulated in all tissues analysed. The retention of the radiotracer at 60-min postinjection was relatively low (<5% ID g−1) in lung, heart, brain, and bone; intermediate (5–8% ID g−1) in plasma, liver, spleen, small intestines, and muscle; and high (>8% ID g−1) in kidney, bile, and urine. The highest [18F]FETA accumulation was found in the excretory organs. Good accumulation in tumours, 6–10% ID g−1, would suggest the possibility of achieving high tumour-to-background contrast in several tumour types, including breast, lung, head and neck, and brain tumours. Chromatographic analysis of plasma, urine, and tissues provided some explanation for the differences in tissue distribution. [18F]FETA was found to be stable in plasma (∼93% as parent compound at 60 min p.i.) and also excreted mainly as the unchanged drug in urine (74% at 60 min p.i.). The radiotracer was metabolised in liver to catabolite(s), which appeared to be eliminated predominantly via the hepatobiliary route.[18F]FETA is taken up into all tissues, we investigated whether differences in pO2 could be monitored with the radiotracer in vivo. Two validation scenarios were explored. In the first instance, the 60-min retention of [18F]FETA into all tumour types as determined by γ-counting was compared to measured pO2. Radiotracer retention positively correlated with the percentage of pO2 values <5 mmHg , indicating that [18F]FETA retention is related to pO2 levels. Using five different tumour types enabled a wide range of oxygenation levels to be achieved , and the relative frequency of pO2 values <2.5 mmHg in a range from 51±13 to 100±0% (P=0.057)). In general, the binding of nitroimidazoles requires the presence of nitroreductases, particularly cytochrome P450 reductase. We found no relationship between [18F]FETA and levels of the nitroreductases studied. Large differences in nitroreductase levels can confound measurement of hypoxia with 2-nitroimidazole-based probes; thus, our findings of a lack of significant differences in nitroreductase activities between the tumour types used is a relevant contribution to the interpretation of [18F]FETA data. The range of nitroreductase levels observed here, ⩽2.5-fold, is unlikely to influence radiotracer retention due to a square-root relationship between the binding of 2-nitroimidazoles and cytochrome P450 reductase levels (18F]FETA binding by oxygen is fairly similar (up to two-fold) across cell lines in vitro. This observation may be explained by the small differences in nitroreductase levels between cell lines. In the second instance, we investigated the ability of [18F]FETA to detect acute changes in tumour oxygenation. Carbogen breathing decreased the 60-min tumour retention of [18F]FETA by 36%, in keeping with an 11% decrease of hypoxic pO2 values measured by OxyLite electrodes. This finding is similar to those reported previously for other 2-nitroimidazoles  showed lower fractional retention of the radiotracer compared to that with the lower VEGF-expression (HT1080/1-3C) despite a higher delivery of [18F]FETA into the former. This finding is in contradiction to the current dogma of ‘hypoxia inducing VEGF through the expression of HIF-1α’  in the HT1080/26.6 model. The impact of these findings on radiation sensitivity was that the HT1080/26.6 tumours were more sensitive to radiation than the HT1080/1-3C tumours as demonstrated by a lower clonogenic radiobiological hypoxic fraction of the former. Since the pO2  of the human tumour models used in this study have not been previously reported in the literature, an assessment of this  was made; the high and low pO2 values obtained for EMT6 and RIF-1 tumours, respectively  dynamically with PET at early time points are attractive features that support the clinical development of [18F]FETA.In summary we have validated an advanced-generation 2-nitroimidazole marker of tumour hypoxia in the mouse. Our findings indicate that [


INK4a constitutes a key event in the multistep process of pancreatic ductal cell transformation. However, the significance of p16 inactivation for complex and tissue-specific aspects of pancreatic cancer progression, such as angiogenesis and metastasis, is less understood. Here, we inducibly re-expressed p16 in vivo in an orthotopic model of pancreatic cancer and examined the impact on these clinically relevant aspects of pancreatic cancer tumour biology. Consistent with previous work in subcutaneous xenograft models, we found p16 capable of reducing primary tumour growth. In addition, p16 restitution resulted in a marked reduction of tumour angiogenesis, largely accounted for by a p16-dependent inhibition of lymphangiogenesis. In excellent agreement with the antilymphangiogenic effect, re-expression of p16 almost completely prevented lymph node metastases of MiaPaca-2 pancreatic tumours. To our knowledge, this is the first report that experimentally links the tumour suppressor p16 to the process of lymphangiogenesis.Functional inactivation of the tumour suppressor protein p16 INK4a, and inactivation of the tumour suppressors p53 and/or DPC4. Most pancreatic cancer cells harbour a combination of 3–4 of the alterations  at 37°C in 95% air and 5% CO2.MiaPaCa-2 cells  were grown as subconfluent monolayers in DMEM  containing 10% FCS, 2 mmol lμg ml−1). TetR promoter activation was achieved with 1 μg ml−1 Doxycycline  and responsiveness was evaluated by transient transfection with pcDNA4/TO-Luc followed by a luciferase assay . The full length cDNA of p16 was subcloned into pcDNA4/TO using restriction enzymes HindIII and KpnI , resulting in pcDNA4/TO-p16. MiaPaCa-2-TREx-p16 cells were obtained through transfection of pcDNA4/TO-p16 into the MiaPaCa-2-TREx cells and selection with zeozin (0.5 mg ml−1)/blasticidin (5 μg ml−1) .The components of the TREx system  include the regulatory plasmid (pcDNA6/TR), the inducible plasmid (pcDNA4/TO) and a luciferase encoding reporter plasmid (pcDNA4/TO-Luc). MiaPaCa-2 cells were transfected  with pcDNA6/TR and stable clones (MiaPaCa-2-TREx) were selected by blasticidin (5 INK4a (1 : 1000) BD Pharmingen, Heidelberg, Germany), anti-pRB (1 : 1000) BD Pharmingen), anti-VEGF-C  or VEGF-D  and the manufacturer's instructions were followed to complete the assay  supplemented with 27% fetal bovine serum . Cells were plated in triplicate on 3-cm dishes and incubated for 10 days. Colonies containing >20 cells were counted with an inverted light microscope.For evaluation of anchorage-independent growth, 10in vivo procedures were in compliance with the UKCCCR guidelines.Female NMRI nude mice were purchased from Taconic M&B, Denmark. The studies were performed in accordance with a protocol approved by the regional animal research committee the ‘Landesamt für Gesundheit und Soziales Berlin Nr. A0136/01’. The −1) and xylazine (20 mg kg−1). In 2 × 12 animals, 1 × 106 MiaPaca-TREx-p16 cells were injected into the head of the pancreas to visibly infiltrate the pancreatic tissue (n=22) p16 induction was started one day after tumour cell inoculation by adding doxycycline (2 mg ml−1) plus 2% sucrose to the drinking water (n=11 mice), whereas control animals received 2% sucrose alone (n=11 mice). All animals tolerated the procedure well. After implantation, mice were inspected daily for body weight loss, general condition, and tumour formation in the peritoneal cavity . At the time of killing, primary tumours were removed and weighted. Tumour volume was calculated using the formula length × width × depth × π/6. Mesenterial and liver hilus lymph nodes were routinely collected for further examination.Orthotopic implantation was carried out when mice were about 7–8 weeks old. General anaesthesia was performed by intraperitoneal injection of a mixture of ketamine , treated with 0.3% H2O2 for 10 min and incubated overnight at 4°C with anti-human p16INK4a  or 1 h at RT with antibodies to mouse LYVE-1 , mouse CD31  or human pan-cytokeratin-HRP  (1 : 50) followed by incubation with a biotinylated secondary antibody. Immunoreactivity was detected with an avidin–biotin complex method (Vectastain Elite ABC kit) and AEC substrate. Sections were counterstained with hematoxylin.Primary tumours and lymph nodes were analysed by standard immunoperoxidase staining procedure. Briefly, 7 For quantitation of microvessel density (MVD) and lymphatic vessel density, respectively, the average numbers of CD31 and LYVE-1 positive vessels from three areas of maximal vascular density (vascular hotspots) were counted .t-test and Fishers exact test using Graph Pad statistical software . Differences were considered significant at P<0.05.Data were analysed by in vivo, a modified tetracycline-inducible expression system . A clone with approximately 10-fold induction (clone MiaPaCa-TREx-10) was identified and utilised in a second transfection to introduce pcDNA4/TO-p16, which encodes p16 under the control of a tet-responsive promoter, such that tetracycline or doxycycline induce p16 expression. Western Blot analysis of multiple independent stable transfected clones revealed variable responses to doxycycline, ranging from no effect (clone number 11) to high expression of p16 associated with varying degrees of promoter leakage .As we intended to study the inducible MiaPaCa-2-TREx-p16 cell system in the orthotopic context, we initially had to confirm the feasibility of this approach. Indeed, MiaPaca-2-TREx gave rise to extensive local tumour growth with invasion of adjacent organs exactly as parental MiaPaca-2 cells. Furthermore, Dox treatment of mice carrying MiaPaca-2-TREx cells was well tolerated and did not significantly reduce primary tumour weight, suggesting that Dox treatment in vivo induction was evident in 6 out of 11 mice with MiaPaCa-2-TREx-p16 orthotopic tumours  and treatment groups (+Dox) at the time of autopsy are presented in  tumours . p16 was tumours . Furthervs 51, P=0.0123, per se did not correlate with MVD  or without lymph node infiltration (N−) . A signiin vivo in the orthotopic tissue context. Consistent with previous work in subcutaneous xenograft models , suggesting p16-controlled angiogenesis through alternate mechanisms.The p16-mediated suppression of the angiogenic phenotype observed here is in excellent agreement with data obtained by inactivation of p16 in the MIN  mouse model of colon carcinogenesis . ColonicOne key contribution to the overall reduction of MVD was the marked decrease of lympahtic vessel density. This experimental link between p16 and lymphangiogenesis provides a pathobiological concept for earlier clinical observations that associated the loss of p16 with lymph node metastasis in several types of carcinomas  p16 significantly inhibited lymph node metastasis, and (ii) mice with lymph node infiltration had significantly higher LVDs in their primary tumours. Given the importance of lymphatic metastasis for the clinical management of pancreatic cancer, our observations provide an important insight into the molecular events that determine this process. They furthermore suggest that p16 mimetic therapies provide benefits beyond the reduction of tumour cell proliferation and primary tumour burden.


In this issue, Length bias may partly explain this phenomenon, in that the smaller screen-detected tumours are more likely to be slow-growing cases than the larger. However, this is unlikely to explain an interaction of the magnitude observed here. The authors conclude that this is evidence that the biological aggressiveness of a breast tumour increases as the tumour develops, notably during its subclinical phases. This is consistent with evidence that histological grade may deteriorate with tumour development, at least in some tumours (et al can also be expressed in terms of the effect of size on node status within screen- and clinically detected tumours separately. In clinically detected tumours, the odds ratio of node positive disease for tumours size 28 mm or more compared to those of size 2–7 mm is 9.6. In screen-detected tumours, the corresponding odds ratio is approximately double this figure at 18.9. Thus, the dependence of node status on tumour size is much stronger in screen-detected tumours.The results of Bucchi The results will surely prompt others to look again at their own tumour data. Some time ago, et al show that the opportunity for screening to lead to detection and treatment before spread to the lymph nodes is extremely limited once the tumour has grown to 20 mm or more in diameter.The practical implication for those involved in breast cancer screening is to further emphasise the importance of detecting the tumour while it is small. Although node status is clearly an important determinant of final outcome (et al shows that for the most part, we do not have continuous tumour size data, but discrete scoring of the underlying continuous phenomenon of size. In such a case, it makes sense to categorise the data around the discrete values recorded.Finally, there is another very interesting aspect to this paper, namely the categorisation of tumour size. Noting the tendency to render this as discrete multiples of 5 mm, the authors chose the categories as 2–7, 8–12 mm, and so on, so that each class represents a cluster around 5, 10 and higher multiples of five. This is a brave but eminently sensible departure from the convention of choosing cutoffs at multiples of 5 or 10 mm, which brings with it the decision whether to include the cutoff point in the lower group, as in the TNM convention or in the upper group, as practised by some researchers. An alternative to categorisation is to use individual values of tumour size as continuous measures, with spline or fractional polynomial regression analyses (


An immunoadsorbent technique is described whereby tumour-specific antibodies may be isolated. Extracts from normal human lung tissue were pooled and bound to cyanogen bromide activated Sepharose 4B. Antisera raised in rabbits to a variety of extracts from human bronchogenic carcinoma were passed through these immunoadsorbent columns to yield antisera specific for tumour-associated antigens as demonstrated by immunodiffusion and immunoelectrophoresis.


Photofrin accumulation in malignant and host cell populations of various tumours was studied by flow cytometry analysis of cells dissociated from the tumour tissue. The transplantable mouse tumour models included in this analysis were sarcomas EMT6, RIF, KHT and FsaN, Lewis lung carcinoma, SCCVII squamous cell carcinoma (SCC) and slowly growing moderately differentiated AT17 SCC. An example of spontaneous mouse adenocarcinoma was also examined. Staining with specific monoclonal antibodies was used to identify the various cell populations present in these tumours. The main characteristic of Photofrin cellular accumulation was a very high photosensitiser content found exclusively in a subpopulation of tumour-associated macrophages (TAMs). Photosensitiser levels similar to or lower than in malignant cells were observed in the remaining TAMs and other tumour-infiltrating host cells. Photofrin accumulation in malignant cells was not equal in all tumour models, but may have been affected by tumour blood perfusion/vascularisation. Results consistent with the above findings were obtained with SCC of buccal mucosa induced by 9,10-dimethyl-1,2-benzanthracene in Syrian hamsters. The TAM subpopulation that accumulates by far the highest cellular Photofrin levels in tumours is suggested to be responsible for the tumour-localised photosensitiser fluorescence.


The tumour-host interaction at the invasive front of colorectal cancer, including the epithelial–mesenchymal transition and its hallmark ‘tumour budding’, is an important area of investigation in terms of prognosis. The aim of this study was to determine the prognostic impact of a ‘pro-/anti-tumour’ approach defined by an established ‘pro-tumour’ (tumour budding) and host-related ‘anti-tumour’ factor of the adaptive immunological microenvironment (CD8+ lymphocytes).n=279; Cohort 1) and immunohistochemistry for CD8+ using tissue microarrays  was carried out. Tumour buds, CD8+ and CD8+ T-lymphocytes : tumour buds indices were evaluated per high-power field.Double immunostaining for CK22/CD8 on whole tissue sections (P<0.001), vascular invasion (P=0.009), worse survival in univariate (P<0.001) and multivariable (P<0.001) analysis, and furthermore in lymph node-negative patients (P=0.002). In Cohort 2, the CD8+/ buds index was associated with T stage (P<0.001), N stage (P=0.041), vascular invasion (P=0.005) and survival in patients with TNM stage II (P=0.019), stage III (P=0.004), and adjuvantly untreated (P=0.009) and treated patients (P<0.001).In Cohort 1, a low-CD8+/ buds index was associated with lymph node metastasis (The CD8+ lymphocyte : tumour-budding index is an independent prognostic factor in colorectal cancer and a promising approach for a future prognostic score for patients with this disease. During the Napoleonic Wars (1796–1815), one of the most effective methods to attack in battle was the deployment of heavy cavalry, which the opponent tried to repel by organizing the infantry into squares.A similar picture can be observed at the invasion front of colorectal cancer. On one hand, the tumour mass invades the pericolic fat tissue, detaching clusters or single tumour cells (tumour buds) reflecting tumour progression; whereas, on the other hand the host attempts to confront this situation by building an ‘anti-tumour’ cytotoxic inflammatory response. This ‘pro-/anti-tumour’ model is supported by a range of studies proposing either independent tumour-related prognostic factors (pro-tumour), such as tumour grade, tumour border configuration, medullary subtype, CEA level, microsatellite instability, loss of heterozygosity (LOH) 18q status, p53 levels, TGFB1 type II receptor levels, VEGF expression, proliferation rate and metalloproteinase expression  or anti-Consequently, several groups focus on the invasive front of colorectal cancer using the term epithelial–mesenchymal transition (EMT), which characterises tumour invasion by de-differentiated colorectal carcinoma cells  and microsatellite instability-high (MSI-H)) seems to relate highly to the number of CD8+ lymphocytes. Compared with MSS tumours, MSI-H cancers are characterised by prolonged survival time, significantly more frequent peritumoural lymphocytic infiltration at the invasive front and by an inherent abundance of intra-epithelial TILs  score, which encompasses information on mitoses, tubule differentiation and nuclear pleomorphism, and is used as an important prognostic feature additional to TNM staging.n=300) and the tissue microarray technique (n=221).Therefore, the aim of this study was to investigate on a potential ‘pro-/anti-tumour’ model and to test the prognostic impact of a ratio defined by an established pro-tumour (tumour budding) and anti-tumour (CD8+ lymphocytes) factor. To this end, two independent colorectal cancer patient cohorts from different centres were investigated using two different approaches, namely whole tissue sections  and pan-cytokeratin  was carried out on one representative slide cut at 4 is study . Double Tumour budding was defined as an isolated single cancer cell or a cluster of up to five cells at the invasive front of colorectal cancer . The tumOf these 300 cases, 279 were evaluable for CD8 and CK22 protein expression, simultaneously. Hematoxylin and eosin (H&E) stained slides were reviewed and histomorphological data included histological subtype, pT stage, pN stage and tumour grade. The tumour border configuration and the presence of conspicuous peritumoural lymphocytic infiltration were defined according to KRAS  and BRAF  were amplified by a first and a nested PCR. Residual primers were removed using the EXOSAPit . Samples were then subjected to direct sequencing of single-stranded PCR products using the BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems) and the ABI Prism 3130 genetic analyser (Applied Biosystems). All products were sequenced bi-directionally. Analysis of MSI status was based on the multiplex amplification of the five microsatellites . An initial denaturation step at 95°C for 10 min was followed by 42 cycles at 95°C for 40 s, 54°C for 40 s and 72°C for 60 s. For the analysis, 1 μl of the DNA weight marker ROX 500 (Applied Biosystem) was added and 10 μl of deionised formamide in 3 μl of the PCR amplified solution. DNA was denaturated by incubation for 2 min at 95°C. The POP-7 polymer solution (Applied Biosystem) was used for the electrophoresis on the ABI Prism 3130 genetic analyser (Applied Biosystems). MSS and MSI-low (MSI-L) status were defined as instability at zero and one markers, respectively. MSI-H was characterised by the presence of instability in two or more markers  protocol and processed in the Xiril X-100 robot . Briefly, punched tissue was lysed in proteinase K. B-beads and MB2 buffer were added to the cleared lysate and shaken for 5 min at room temperature. The supernatant was removed and MB3 was added followed by shaking and supernatant removal. The genomic DNA was eluted with MB6 buffer. Genomic DNA was amplified by PCR using AmpliTaq Gold polymerase . A tissue microarray of 221 unselected, non-consecutive colorectal cancer patients treated at the Second Department of Pathology, University of Athens between the years 2004 and 2006 was constructed at the Institute for Pathology, University Hospital of Basel. The use of this material was approved by the local ethics committee of the University of Athens.2) using a homemade semi-automated tissue arrayer. Tissues were obtained from the tumour centre, the invasive tumour front within the representative area of most intense tumour budding in all sections of the tumour, as determined from corresponding H&E slides, the normal adjacent mucosa (if available) and the transitional zone where tumour and normal adjacent mucosa first interact (if available). Each patient on average had 5.1 tissue punches included on this array. The final tissue microarray contained 1079 tissues, namely 437 tissues from the tumour centre, 430 from the invasive front, 90 from normal adjacent mucosa and 122 from the transitional zone. For the purposes of this study, only tissue punches from the invasive tumour front per patient were analysed.Each patient had multiple tissue punches taken from formalin-fixed, paraffin-embedded blocks using a tissue cylinder with a diameter of 0.6 mm, which were subsequently transferred into one recipient paraffin block  at 800°C for 6 min followed by incubation at room temperature for 10 min. After wash steps, peroxidase blocking was carried out for 10 min. Sections were again washed and then incubated with primary antibody against CD8  for 60 min. Subsequently, sections were washed with TBS and incubated with Real Envision Solution (Dako) for 30 min, then washed again in TBS and incubated with DAB-Chromogen for 10 min. After washing, sections were counterstained with haematoxylin for 2 min. Only peritumoural CD8+ T-lymphocytes in punches taken from the invasive tumour front were counted.Immunohistochemistry was carried out using anti-CD8 antibody at the Second Department of Pathology, University of Athens. Briefly, 5-All cut-off scores to classify patients as having a ‘low’ or ‘high’ index were obtained by receiver operating characteristic curve (ROC) analysis  and 95%CI were obtained to determine the prognostic effect of each index after adjustment. With logistic regression analysis, the odds ratios (OR) and 95%CI were obtained to determine the odds of death at 5 years with the use of different indices. All statistical analyses were carried out using SAS .The association of indices with categorical clinico-pathological features was obtained using the P=0.017), with the presence of lymph node metastasis (P=0.002), with the presence of vascular invasion (P=0.028) and with an infiltrating tumour border configuration (P=0.005) (P=0.022), whereas no association was observed with KRAS or BRAF mutation. The odds of death from disease at 5 years in the group with a high-budding index was OR (95%CI)=1.89 (1.1–3.3) compared with those with a low index (P=0.022) (P=0.033) (P=0.019), pN stage (P=0.002), vascular invasion (P=0.004) and an infiltrating tumour border configuration (P=0.021) and worse survival in univariate analysis (P=0.02). In MSI-H patients, the high tumour-budding index was only related to an infiltrating tumour border configuration (P=0.015).A high tumour-budding index was defined, using ROC curve analysis, as at least 16 buds per × 40 field . HoweverP=0.005) . PatientP=0.022) . In all,P=0.033) . In patiP=0.002), with MSI-H status (P=0.033) and marginally with BRAF mutation. Patients with a high CD8+ index demonstrated a significantly prolonged survival time compared with those with a low CD8+ index (P<0.001) (P=0.011).A high CD8+ index was characterised by the presence of at least 40 CD8+ cells per × 40 field . The disP<0.001) . In MSS/vs the number of tumour buds led to a strong discrimination of survivors and non-survivors of colorectal cancer, as indicated by an AUC value of 0.68 (95%CI 0.61–0. 75) (P<0.001), absence of vascular invasion (P=0.009), presence of a pushing/expanding tumour border configuration (P=0.005), presence of peritumoural lymphocytic inflammation at the invasive front (P=0.007) and a marginal association with MSI-H status (P=0.052). Most notably, a considerable difference in survival time between patients with low and high CD8+/ buds index was observed with patients with a low index demonstrating a highly adverse prognosis (P<0.001) (P=0.001).A ratio of at least three times more CD8+ T-cells 1–0. 75) . The odd1–0. 75) . PatientP<0.001) . This prn=226, 101 deaths). The budding index was not found to add independent prognostic information when analysed along with these additional features (P=0.85), whereas the CD8+ index (P<0.001) and the CD8+/ buds index (P<0.001) were highly relevant as prognostic factors in multivariable analysis, with similar relative risks of death suggesting a comparable performance of these two features as prognostic indices.A comparison of the performance of each of these indices in multivariable survival analysis is outlined in n=140, 42 deaths), as well as of those with colon  or rectal cancers . In lymph node-negative patients, multivariable analysis with pT stage, vascular invasion and tumour border configuration revealed independent prognostic effects for both the CD8+ index (P=0.001) and the CD8+/ buds index (P=0.002), but not for the budding index. Moreover, in patients with colon or rectal cancer, the CD8+ index  as well as the CD8+/ buds index  maintained a significant independent prognostic effect after adjusting for pT stage, pN stage and vascular invasion.In order to prevent over-fitting of multivariable models, only the most relevant prognostic factors were included into sub-group analyses of lymph node-negative patients (P=0.029), more poorly differentiated tumours (P=0.003), presence of vascular invasion (P=0.045) and was highly related to a worse prognosis (P=0.003) (P=0.053), early tumour grade (P=0.014), absence of vascular invasion (P=0.002) and a prolonged survival time compared with patients with a low CD8+ index (P=0.031).In Cohort 2, a high tumour-budding index in stages I–III patients was defined as at least six buds per punch and was related to a more advanced pT stage (P=0.003) . The thrP=0.001), lymph node-negative (P=0.055), early tumour grade (P<0.001) and vascular invasion negative (P=0.002). Low CD8+/ buds index, led to a highly unfavourable prognosis compared with patients with a high CD8+/ buds index (P<0.001). This difference was also maintained in sub-group analysis of TNM stage II (P=0.019) and stage III (P=0.004) patients   . MultivaThe aim of this study was to investigate an alternative approach to reflect the dynamic at the tumour front of colorectal cancer using an index including tumour budding and CD8+ lymphocytes.Briefly summarised, our results confirm the prognostic impact of the selected tumour and host-related factors. In both patient cohorts, a high tumour budding and a low CD8+ lymphocytes index were associated with tumour progression and worse survival. Indeed, tumour budding has previously been found to be associated with higher N stage, higher tumour grade and presence of vascular invasion, local tumour recurrence, distant metastasis as well as worse survival (The question, therefore, arises as to whether a CD8+ lymphocytes : budding index is more advantageous over the use of one out of the two parameters alone. With the exception of 5-year survival, a certain intra- and inter-group variability is observed when analysing the relationship of tumour budding or CD8+ lymphocytes with clinico-pathological parameters. In contrast, the use of a CD8+ lymphocytes : budding index better summarises these associations with key endpoints of tumour progression, namely presence of lymph node metastases, vascular invasion and worse survival, and moreover, in both cohorts. In addition, the CD8+/ buds index shows an increased discriminatory ability for identifying survivors and non-survivors at 5-year follow up compared with tumour budding or CD8+ lymphocytes count themselves. More specifically, patients with a low CD8+ lymphocyte : buds index have more than four-fold odds of death at 5 years compared with a three-fold or under two-fold risk for patients with low CD8+ counts or high number of tumour buds, respectively. In addition, the CD8+/ buds index maintained its significant impact on outcome in patients with stage II disease, in both colon and rectal cancer patients and also, independently of adjuvant therapy. Our previous work describes the absence of tumour budding in cases with marked peritumoural inflammatory reaction at the invasive margin of colorectal cancers and a strong correlation between this inflammation and the presence of TILs(Several points in this study should still be considered. Although the impact of tumour budding on clinical outcome is validated by several groups, a standardised scoring system for this feature has not yet been established, although several similar methods have been proposed . This stOn the other hand, several factors strongly support the validity of our findings. The selection of parameters included in the proposed index is evidence based, as both tumour budding and the CD8+ lymphocytes have been tested and validated in colorectal cancer by different research groups. The index, which encompasses a tumour- and host-related feature, and therefore represents the more advantageous ‘multi-marker’ approach to prognosis, better reflects the tumour dynamic at the invasive front, compared with either feature alone. This study also benefits from the inclusion of two completely independent cohorts of colorectal cancer patients treated in Switzerland and Greece, respectively. In addition to whole tissue sections, we used the tissue microarray technique, a powerful tool for the investigation of novel or potential biomarkers, which has allowed us to evaluate hundreds of tissue cores for CD8+. Moreover, the evaluation of multiple tissue punches per tumour in this study exceeds the number of cores required for adequate representativity of tumour heterogeneity (In conclusion, the CD8+ lymphocyte to tumour-budding index is an independent prognostic factor in colorectal cancer and represents biologically a ‘pro-/anti-tumour’ model that could be a promising approach for a future prognostic score in colorectal cancer.