Prostate cancer is a frequent finding in man. In dogs, malignant disease of the prostate is also of clinical relevance, although it is a less common diagnosis. Even though there are numerous differences in origin and development of the disease, man and dog share many similarities in the pathological presentation. For this reason, the dog might be a useful animal model for prostate malignancies in man.in vivo testing, and to distinguish the growth pattern of the induced tumours.Although prostate cancer is of great importance in veterinary medicine as well as in comparative medicine, there are only few cell lines available. Thus, it was the aim of the present study to determine whether the formerly established prostate carcinoma cell line CT1258 is a suitable tool for in vivo behaviour of the in vitro established canine prostate carcinoma cell line CT1258, cells were inoculated in 19 NOD.CB17-PrkdcScid/J (in the following: NOD-Scid) mice, either subcutaneously or intraperitoneally. After sacrifice, the obtained specimens were examined histologically and compared to the pattern of the original tumour in the donor.For characterisation of the Cytogenetic investigation was performed.The cell line CT 1258 not only showed to be highly tumourigenic after subcutaneous as well as intraperitoneal inoculation, but also mimicked the behaviour of the original tumour.in vivo model for future studies.Tumours induced by inoculation of the cell line CT1258 resemble the situation in naturally occurring prostate carcinoma in the dog, and thus could be used as  Currently, the dog is used as  lesions -3. Where lesions . Since t lesions ,5. AlthoAlthough there are some reports observing a permissive or protective effect of androgens -10, andrDespite the rather high incidence of prostate carcinoma in man, there are only three cell lines of human prostate carcinoma and their sub lines available. Thus, it is not surprising that there are only occasional reports about cell lines of canine prostatic carcinoma -13. HencThe cell line was derived from a prostatic tumour of a Briard, 10 years, male intact, which had been presented at the Small Animal Clinic of the University of Veterinary Medicine Hanover, Germany. The dog had a two week history of dyschezia, gain of abdominal girth, polydipsia, and loss of appetite. Clinical examination revealed an undulating and strained abdomen. Abdominal radiographs showed a highly reduced perceptibility of detail. On abdominal ultrasound an enlarged prostate could be detected, which contained several cysts.Explorative laparatomy was performed and about 3000 ml abdominal fluid were obtained; the prostate itself was highly enlarged and several miliary masses were found in the mesentery. There was no evidence of contact metastases to abdominal organs or distant metastases to the lung. Biopsies have been taken under general anaesthesia; the cytological diagnosis of a highly malignant adenocarcinoma of the prostate resulted in euthanasia ad tabulam. Prior to euthanasia, biopsies have been taken for further histological examination and for cell culture. Histological staining and immunohistochemical staining as well as cytogenetic analysis was performed as described below.The cell culture conditions, as well as the characteristics of the canine prostate carcinoma cell line CT1258 have been described previously .ad libitum, any manipulation was performed in a laminar flow hood. The animals were observed on a daily basis and sacrificed depending on the clinical condition and the size of detectable tumour, respectively. The tumours were allowed to grow up to a diameter of 10 mm. Additional criteria for euthanasia were ulceration of subcutaneous masses, a reduced level of activity of the mice, and the loss of appetite. The animals were humanely sacrificed by cervical dislocation after inhalation of > 70% carbon dioxide. The study was approved by the Lower Saxony State Office of Consumer Security and Food Safety (33-42502-05/950), the ethical approval was sought from the University of Veterinary Medicine Hanover.This study involved 19 NOD-Scid mice . All animals were bred and maintained in a protected environment at the Institute of Laboratory Animal Science of the Hanover Medical School. The mice were fed autoclaved food and water The cells were harvested from the culture flasks with 1 ml of TrypLE Express  and incubated at room temperature for 1 minute. L-199 medium  supplemented with 20% FCS was added and the cells were centrifuged at 350 g for 15 minutes. The cell pellet was washed twice with PBS. The cells were resuspended in 200 μl PBS; immediately before application the suspension was aspirated into Insulin-Syringes  and inoculated either subcutaneously into the left flank of the animals or intraperitoneally.6 cells. For intraperitoneal application, mice received either 1 × 105 or 5 × 105 cells. The 19 NOD-Scid mice were subdivided into two groups: One group consisted of four female and five male mice, the animals received 1 × 106cells subcutaneously each. The second group consisted of five male and five female animals, cells were inoculated intraperitoneally. Four of them  received 1 × 105 cells; six animals  received 5 × 105 cells, respectively.For subcutaneous inoculation, the animals received 1 × 10in vitro culturing and Cytogenetic analysis. In order to address the growth pattern, the original canine tumour and the murine tumours have been classified based upon the presence of glandular, urothelial, squamoid, or sarcomatoid differentiation.Necropsy was performed immediately after death had occurred. Lung, liver, spleen, kidneys, gonads, bowel, and detected masses were removed, fixed in 10% buffered formalin, paraffin embedded, sectioned, and stained with hematoxylin and eosin; immunohistochemical staining for Ki67 was performed, using the monoclonal antibody MIB-1  in a dilution of 1:100. The paraffin sections were pretreated in a microwave oven for 20 minutes in citrate buffer solution, pH 6. The secondary antibody was a biotinylated goat-α-mouse antibody, for detection, the Vectastain ABC-Kit  has been used. The Ki67 Index was determined by detecting the fraction of Ki67 positive stained cells in total 500 cells. This was performed in the original tumour, as well as in the induced tumours. Intestinal mucosa served as positive control. In addition, tumour samples of each mouse were obtained in liquid nitrogen for real time RT-PCR and in Hank's Medium for further For cytogenetic investigation, cells have been processed as described previously . Tissue This study was intended to be descriptive rather than statistically significant. However, a paired student's t-test was performed. A P-value < 0.05 was considered to be significant.On histological examination the tumour showed to be consisting of poorly differentiated cells with numerous signs of malignancy. A strong anisocytosis, numerous cells with multiple nuclei, anisokaryosis, multiple mitotic figures, to some extent atypical mitotic figures, and a varying nucleus: plasma ratio could be detected. The tumour showed a compact growth with glandular differentiation. The Ki67 index was 43%.6 cells of the canine prostate carcinoma cell line CT1258 (Table For analysis of duration until sacrifice of the mice, only those individuals were considered that were sacrificed due to tumour burden. All female mice and four of five male mice developed a detectable tumour mass after subcutaneous inoculation of 1 × 10Histology revealed a highly heterogeneous population of cells, numerous mitotic figures, anisokaryosis, anisocytosis, and several cells containing more than one nucleus; there was a variable nucleus: plasma ratio Figures , 2. The 5 cells, consisting of two males and two females each, and animals receiving 5 × 105 cells, consisting of three males and three females each. The criteria for sacrification were impairment of the general condition, worsening of the nutritional condition, and/or reduction of activity level. Weight gain was not a reliable factor due to the development of massive peritoneal effusion.The group was subdivided in two categories: Animals receiving 1 × 105 cells tumour growth could be detected. The mean duration until sacrifice was 28 days. In one of three females and two of three males inoculated with 5 × 105 cells tumour growth could be detected, in this group the mean duration until sacrifice was 26 days (Table 5 cells, had an extended abdomen and proved to have peritoneal effusion. The male without obvious tumour growth had developed a thymoma, which was the cause for the bad clinical condition. Six of these ten mice developed a mass at the injection site, although at inoculation a proper amount of time had elapsed until withdrawal of the needle. Necropsy showed a moderate to high tumour burden at the peritoneum and no visible signs of metastasis to the lungs (Figure In all mice receiving 1 × 10s Figure . HistoloThe cytogenetic investigation of intraperitoneal and subcutaneous tumours revealed the same markers that have been shown in analysis of the original tumour and of the cell line . A hyperIdeally a cell line should not only be immortalized spontaneously but also be tumourigenic in experimental animals. Another claim may be the mimicking of the original natural behaviour of the tumour in the host. This condition promotes the predictability of any results obtained with that very cell line. This is true especially for cell lines derived from nonhuman tumours if the origin may serve as a model itself.This study reveals that the cell line CT1258 is highly tumourigenic in NOD-Scid mice. Local tumour growth occurred in 89% of the animals that had received cells subcutaneously; 86% of the animals that had been inoculated intraperitoneally and that had developed tumour growth also developed local tumour growth at the injection site. The latter was not desired, and since it is not known how many cells remained in the branch canal, the size of those accidental masses cannot be compared accurately. Focussing on the mice with cells inoculated i.p., 86% developed peritoneal effusion just as the donor did and all of them had multiple small nodules within the serous membranes of the peritoneum rather than one large mass. The excellent local tumour growth on one hand and the rapid development of pleural effusion on the other hand indicates that in studies to come the use of the cell line might be of a greater value in local administration rather than in systemic inoculation. Orthotopic implantation might be of great value, since this could enable the assessment of local growth and invasion as well as potential metastasis. Due to the lack of vascularisation, the tumour cells found in the lungs of the mice must be addressed as tumour cell embolism rather than metastasis. Since neither the mice nor the donor had evidence of lymphogenic or hematogenic metastasis, we conclude that time for metastasis exceeds the time limitation owed to local tumour growth. Whether this is due to the rapid growth rate or due to potentially little disposition of the cells to degrade extracellular matrix and therefore has a generally low tendency to metastasise remains unclear. The absence of bone metastasis is a remarkable fact, which is contrary to the high incidence of bone metastasis in canine and human patients . Potenti5 cells developed tumours, but 50% of the animals that received 5 × 105 cells did not; although this is not considered to be statistically significant, it suggests that an even lower number of cells might have been sufficient to induce a tumour. The difference between male and female animals is not statistically significant.The number of cells obviously did not seem not to have a direct impact on tumour growth, since all mice that had been inoculated with 1 × 10The Ki67 indices in both, the original tumour and the experimentally induced tumours, were considered to be high. The difference between the original tumour on one hand and the subcutaneous and intraperitoneal masses on the other hand were statistically not significant. In human medicine a high Ki67 index has shown to be associated with a poor prognosis . ConsideOne limiting factor is that two of the nineteen mice in the study developed thymoma within the course of the study. This is a frequent finding in older NOD-Scid mice ,17. TherThe canine prostate carcinoma cell line CT1258 demonstrated to be tumourigenic in the NOD-Scid mouse. Tumours induced in mice resembled the biological behaviour of the tumour from which the cell line was originally derived regarding growth pattern and histological appearance. Therefore we conclude that the use of this animal model will provide results with a high predictability towards clinical use in veterinary medicine and due to the correlation between canine and human prostate carcinoma in humans as well. The lack of skeletal metastasis is a potential field for further studies.The authors declare that they have no competing interests.in vivo work up, JB and IN incited the study and coordinated the operational procedure. All authors read and approved the final manuscript.MAMF was involved in study-design, implemented the in vivo tasks and the histopathology, and wrote the paper, HME was involved in study-design and coordination of in vitro work up, JTS and KAS assisted with dissection of the animals and in vitro work up, SaW prepared the cells for inoculation, SuW was involved in study-design, NR–B was involved in chromosomal preparation, cytogenetic analyses and karyotyping, MD and HHJH were involved in study-design and supervising the The pre-publication history for this paper can be accessed here:


A patient with the midgut carcinoid syndrome with severe diarrhoea and proven hypersecretion of serotonin (5-HT) was treated with low doses of verapamil perorally. During treatment the patient was completely relieved of diarrhoea but discrete facial flushing persisted during treatment. When treatment was cessated, diarrhoeas recurred. This patient underwent pentagastrin (PG) provocation repeatedly; during untreated conditions injection of PG released 5-HT, detectable in peripheral venous blood. Such release was abolished during verapamil treatment, but recurred after withdrawal of the drug. Surgical biopsies from this tumour were studied in two experimental models: cell suspensions and heterotransplants grown in the anterior eye-chamber of immunosuppressed rats. Release of 5-HT from the cell suspensions was elicited in a dose-dependent manner after stimulation with isoprenaline (IP) suggesting activation of beta-adrenoceptors on the tumour cells. Such release was reduced after pretreatment with verapamil indicating a calcium dependent mechanism. Intraocular tumour transplants also responded with release of 5-HT into the chamber fluid after conjunctival application of IP. However, pretreatment of the rats with verapamil significantly reduced the IP-stimulated release of 5-HT.


Young born to and suckled by mothers rendered immune to a syngeneic sarcoma showed no acquired resistance to challenge with tumour. Whilst other workers had found that allograft immunity could be transferred by milk, we conclude (1) that lymphocytes present in the milk cannot transfer immunity to tumour-specific antigens when absorbed by suckling, and (2) that transfer of maternal immunoglobulins either pre- or postnatally also does not protect against s.c. inoculated tumour.


Mdr1a, and levels of circulating IL-6. Malignancy results in reduced hepatic drug disposition that correlates with an associated inflammatory response. Reduction of inflammation may improve the clinical outcome for patients receiving chemotherapeutic agents that undergo hepatic metabolism.There is increasing evidence of a systemic inflammatory response associated with malignancy, which may have an impact on both drug disposition and resistance to cytotoxic therapy. The impact of inflammation on drug disposition was studied in mice bearing a number of common tumour xenografts. C57BL/6 mice were inoculated with tumour xenografts. Hepatic expressions of Cyp3a and drug transporters were analysed at the mRNA, protein and functional levels (Cyp3a only). Circulating serum cytokines and the hepatic expression of acute phase proteins (APPs) were measured. Intratumoral levels of multidrug resistance genes were determined. Tumour xenografts elicited an inflammatory response that coincided with repression in hepatic Cyp3a11 activity and the expression of a number of hepatic drug transporters. With tumour growth, a progressive reduction in hepatic Cyp3a11 mRNA expression was seen. Conversely, an increase in the hepatic APP expression and circulating interleukin (IL)-6 levels was observed. Furthermore, a correlation was seen between increased intratumoral expression of the multidrug resistance gene,  The pharmacokinetics (PKs) of chemotherapeutic drugs vary considerably between patients, and because of the narrow therapeutic index of most anticancer agents, results in their inherent lack of safety. Variability in the PKs of anticancer agents can be attributed to a number of factors including comedications, environmental factors, genetic polymorphisms and certain disease states. These factors can have an impact on drug disposition, resulting in either ineffective drug doses or excessive toxicity . This coThe presence of a tumour-induced inflammatory response is increasingly being recognised in patients with solid tumours .P450 activity, in particular CYP3A4, has been extensively studied both in vitro and in vivo (β and tumour necrosis factor-α (TNF-α), resulting in reduced drug metabolism and clearance (The relationship between inflammation and repression of +-taurocholate cotransporting polypeptide (NTCP) and organic anion-transporting polypeptide (OATP) families. Following metabolism, efflux of metabolites across the canalicular surface of the hepatocyte into bile is facilitated by members of the ABC family of transporters, specifically the bile salt export pump (Bsep), multidrug resistance-associated protein (MRP2) and members of the multidrug resistance (MDR) family. Efflux across the basolateral surface of the hepatocyte back into the circulation is mediated by MRP3 and MRP4  or 3.8 × 106 E0771 breast cancer cells  suspended in 0.3 ml of PBS subcutaneously into the right flank. Control animals were inoculated with the vehicle alone.Eight- to ten-week-old male mice were aseptically inoculated with either 0.3 ml suspension of Englebreth-Holm-Swarm (EHS) sarcoma into the right quadriceps muscle, 3.8 × 103) was calculated using the equation volume=0.5 × (width × breadth × height). Tumour mass reached approximately 3 g or 10% of total body weight after approximately 3 weeks. Mice were anaesthetised with ketamine and xylazine  before cardiac puncture for the collection of blood. The liver was immediately harvested and snap-frozen in liquid nitrogen and stored at −70°C. Serum was snap-frozen in aliquots in liquid nitrogen and then stored at −70°C. For the time-course experiments, mice inoculated with the EHS sarcoma were harvested at four different time points: days 4, 7, 11 and 18.Mice were weighed and calliper measurements of the subcutaneous tumour were made daily. The volume of tumour  using random hexamer primers. Taqman or SYBR green protocols were used to amplify cDNAs of interest by real-time quantitative PCR (Q-PCR) using a Rotor-Gene 3000 . Primers were obtained from Invitrogen and primer/Taqman probes from Applied Biosystems . Messenger RNA levels were normalised to 18S mRNA expression. Normalisation to 18S, B2M and C36B4 mRNA housekeeping genes gave comparable results. Primer and probe sequences are available on request.Total RNA was isolated from mouse liver and tumour using Trizol reagent  and treated with DNase I  according to the manufacturer's protocols. Complementary DNA was synthesized from 5 μg of protein was loaded and resolved on 7% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) for 2 h under reducing conditions and then transferred to polyvinylidine difluoride membranes. Membranes containing transporter proteins were then cut into half and the upper portion (mol. wt. >72 kDa) was incubated with either cMOAT/MRP2 (clone M2III6) antibody  or MRP3 (clone M3II-9) antibody (Signet), followed by a peroxidase-conjugated secondary antibody . To control for variability in protein loading, the lower portion of the membrane (mol. wt. <72 kDa) was incubated with the anti-β-actin clone AC15 antibody (Sigma-Aldrich), followed by the secondary antibody. Mouse Cyp3a proteins were detected with a polyclonal rabbit anti-rat Cyp3a antibody . Proteins were visualised by SuperSignal West Pico chemiluminescence kit  and quantified using densitometric analysis.Extraction and preparation of reduced proteins from liver tissue were performed as described previously . Mice were deemed to be asleep when loss of righting reflex was observed, and awake when this reflex returned.To determine whether the transcriptional repression of murine β, TNF-α and IL-6 immunoassays as per the manufacturer's instructions .Cytokine levels in the serum of tumour-bearing and control mice were measured using the Quantikine high-sensitivity mouse IL-1n⩾4 mice. Differences between tumour-bearing and non-tumour-bearing mice were determined by one-way analysis of variance and logistic regression analysis, with P<0.05 considered to be statistically significant. Data were log-transformed before analysis in order to stabilise variance if deemed necessary. Analysis was performed using SPSS version 11.5 .All studies were performed using groups of 3 (2216–3596 mm3); breast E0771, 2177 mm3 (228–7399 mm3); and EHS sarcoma, 4690 mm3 (3188–6459 mm3).To determine whether the presence of extra-hepatic malignancy affects the activity of hepatic Cyp3a11, mice were injected with a number of tumour xenografts or vehicle. Following tumour inoculation, mice maintained good health and activity levels with no net weight loss observed. No adverse events were noted during the study period and no mice died during the experiment. The time to harvest from tumour implantation was as follows melanoma B16, 20 days; breast E0771, 16 days; and EHS sarcoma, 24 days. The average tumour volume of each tumour type at harvest was as follows melanoma B16, 2290 mmOn Q-PCR, the presence of malignancy resulted in a significant repression in hepatic Cyp3a11 mRNA compared with controls for all tumour types . These rP=0.08) and breast cancer (P=0.06) xenografts compared with controls. The proinflammatory cytokines IL-1β, TNF-α and IL-6 were quantified using ELISA. No significant differences were observed in the circulating levels of IL-1β and TNF-α between control and tumour-bearing mice (data not shown). However, the level of IL-6 was significantly elevated in all tumour models compared with controls .In order to assess whether the repression of hepatic Cyp3a11 mRNA expression and the presence of inflammation were dependent on tumour growth or the presence of malignancy ing mice . A signiing mice . Furthering mice . A statiin vitro induces drug resistance, the expression of multidrug resistance genes was assessed within the tumour at different time points  and mRNA expression levels of IL-6 within the tumour itself . Combined with the observed decrease in expression of MRPs and other ABC transporters in the livers of tumour-bearing mice (As administration of exogenous IL-6 to cancer cells e points . This waing mice , this fiAlterations in the expression of enzymes and transport proteins involved in hepatic drug disposition can have an important influence on the susceptibility of organs and tissues to the therapeutic and toxic effects of anticancer drugs. The first aim of this study, therefore, was to assess the effect of extra-hepatic malignancy on hepatic Cyp3a mRNA expression and activity, and whether this was associated with a tumour-induced acute phase response.We have shown in murine models of a number of common malignancies that there is a significant repression of hepatic Cyp3a11 mRNA expression in tumour-bearing mice compared with controls, functionally reflected by a prolongation of the sedative effect of midazolam, a CYP3A substrate. This study further expands on previous experiments from our group in which a transgenic mouse model of human CYP3A4 regulation was used to demonstrate that the presence of tumour resulted in downregulation of the CYP3A4 transgene, which was linked to a systemic acute phase response. Furthermore, IL-6 was localised to the tumour and not the liver in tumour-bearing animals, suggesting that the tumour or its associated stroma is the source of IL-6. As a result of this study, a mechanistic link was suggested between tumour-derived cytokines and impaired drug metabolism (in vitro and in vivo. In vitro studies in hepatoma cell lines illustrate the downregulation of Cyp3a11 exposed to the proinflammatory cytokines IL-6, IL-1β and TNF-α (β, TNF-α and especially IL-6 were observed (in vivo studies in IL-6-deficient mice, no repression in Cyp3a11 was seen on treatment with turpentine or tuberculosis vaccine (β and TNF-α receptors in vivo had little or no effect on Cyp3a11 downregulation (While the cytokines contributing to the systemic inflammatory response associated with malignancy have not been extensively characterised, the cytokines involved in the repression of murine Cyp3a11 in acute inflammation have been extensively studied both in vitro and in vivo models of acute inflammation in which IL-6 is reported to be the major contributor to the repression of hepatic transporter mRNA expression (The next aim of this study was to assess the impact of tumour-associated inflammation on the expression of hepatic transporters in order to ascertain whether derangement of drug disposition represents a more global phenomena occurring within the liver, involving more than drug metabolism. This is the first report of the involvement of drug transporters in the altered drug response in malignancy, specifically, the transcriptional downregulation of the hepatic mRNA expression of Mdr2, Mrp2, Mrp3, Ntcp, Oatp2, Oatp-c and Bcrp. This represents a widespread repression of drug transport within the liver with transporters mediating both uptake and efflux from the affected hepatocyte. These findings are consistent with previously reported pression . The mRNpression . We did pression . These fin vitro studies in which the autocrine production of IL-6 in breast cancer cells was demonstrated to increase MDR expression within the tumor cells themselves (The final aspect of this study was to determine the effect of tumour size on Cyp3a expression and plasma cytokine concentrations. We have clearly shown that the transcriptional downregulation of Cyp3a11 mRNA is dependent on tumour growth. This was also true of the acute phase response with progressive increases in hepatic Sap mRNA and levels of circulating IL-6 observed at each advancing time point. Furthermore, we observed an increase in the expression of Mdr1a within the tumour of mice bearing the EHS sarcoma from day 11 to day 18. This was in direct contrast with the mRNA expression of Mdr1a within the liver that is reduced, albeit not significantly. These time-course studies are novel in that they illustrate that the acute phase response and subsequent impaired drug metabolism are dependent on tumour growth. This suggests that the tumour itself, its supporting vasculature and/or stroma may be the source of IL-6. Furthermore, our findings suggest an association between circulating IL-6 levels and the upregulation of multidrug resistance genes within the tumour. This is consistent with per se, as they remained in good health throughout the experiment. We have shown that tumor-bearing animals have evidence of a systemic inflammatory state and that this contributes to a global derangement of hepatic drug handling. Furthermore, we have shown that this is not specific for a particular tumour type but is a general phenomenon that applies in varying degrees to a number of tumour types. This is in the context of increased multidrug resistance gene expression within the tumour itself. While the molecular mechanisms are not well understood, this study raises the possibility that patients with cancer who develop an inflammatory response may have reduced metabolism and tumour chemosensitivity and/or increased toxicity. Serum concentrations of APPs or cytokines could potentially be used as predictive factors for hepatic handling of cytotoxic drugs. In addition, reversal of the inflammatory process before administration of chemotherapy could potentially negate the deranged drug phenotype and improve treatment tolerance and efficacy. These animal models create an experimental platform to assess such reversal strategies that could guide future clinical studies.The findings of the present study are unlikely to be the result of the mice being perturbed from the tumour implantation in vivo. Further work is also needed to investigate the interplay of cytokine signalling pathways with transcription factors known to be relevant to drug-metabolising and transporting genes such as hepatocyte nuclear factor-4α, signal transducer and activators of transcription 3, CCAAT enhancer binding proteins and the pregnane X receptor, in order to further understand the mechanism by which IL-6 regulates drug clearance in the presence of tumours (Additional studies are required to ascertain whether a correlation exists between reduced expression of hepatic transporters and impaired drug handling


Mice transplanted with NC carcinoma were treated with the thromboxane synthetase inhibitor dazmegrel (UK38485) or with nafazatrom (BAY G 6575), a compound that is reported to increase prostacyclin formation. Some experiments included the cytotoxic drugs methotrexate and melphalan. The tumours were excised under anaesthesia on day 14 or day 21 after transplantation, and weighed; some were extracted for prostanoids which were measured by radioimmunoassay. Mouse survival time was determined up to day 121, and cancer spread was determined by postmortem examination. The survival was increased by methotrexate and melphalan but not by the other drugs. Nafazatrom-treated mice tended to have lighter tumours. Although dazmegrel reduced the formation of thromboxane B2 during clotting of blood from normal mice, it did not affect the tumour yields of prostanoids. Nafazatrom had no effect on serum or tumour prostanoids. There were no obvious effects of the treatments on the recurrence of tumour in the excision scar, lung metastasis or spread to lymph nodes.


We show here, using high-resolution magnetic resonance imaging, that injured tissue provides a favourable milieu for the neovascularization and growth of C6 glioma spheroids, implanted subcutaneously in nude mice. Moreover, the presence of micro-tumours in an injured tissue inhibited the healing process, leaving an open persistent wound. In correlation with the induced angiogenesis of implanted spheroids in the presence of proximal wounds, a shorter lag period was observed for initiation of tumour growth. This effect was restricted spatially and was observed only for wounds within 5 mm from the tumour. In such proximal wounds, angiogenesis was enhanced in the first days after injury, and vessel regression, which normally starts 4 days after injury, did not occur. Injury causing interference to tumour perfusion promoted tumour vascularization and growth even for more remote incisions, possibly by activating stress-induced angiogenesis. The kinetics of vascularization and growth of these wound-tumour systems sheds light on the clinical observations of increased probability of metastatic recurrence and stimulated regrowth of residual tumour in the site of surgical intervention. High-resolution magnetic resonance imaging could detect the aberrant angiogenic activity of these tumour-wound systems as early as 1 week after injury.


Serial transplantation of an HCG-producing human testicular teratoma in immune-deprived mice is described. The xenografted tumour was compared to the tumour of origin in histology, immunohistochemistry  autoradiography, marker production and growth rate. It is concluded that the xenograft retained the characteristics of the original tumour with the exception of a reduction in HCG-producing elements at transplantation beyond 5 serial passages.


O-linked disaccharide that can be carried on various glycoproteins. One such glycoprotein MUC1 is expressed by the vast majority of breast carcinomas. Both STn and MUC1 have been considered as targets for immunotherapy of breast cancer patients. Here we used different immunogens to target STn in an MUC1 transgenic mouse model of tumour challenge. We show that synthetic STn coupled to keyhole limpet haemocyanin (Theratope), induced antibodies to STn that recognised the glycan carried on a number of glycoproteins and in these mice a significant delay in tumour growth was observed. The protection was dependant on STn being expressed by the tumour and was antibody mediated. Affinity chromatography of the STn-expressing tumour cell line, followed by mass spectrometry, identified osteopontin as a novel STn-carrying glycoprotein which was highly expressed by the tumours. These results suggest that if antibodies can be induced to a number of targets expressed by the tumour cells, a humoral response can be effective in controlling tumour growth.Changes in the composition of glycans added to glycoproteins and glycolipids are characteristic of the change to malignancy. Sialyl-Tn (STn) is expressed by 25–30% of breast carcinomas but its expression on normal tissue is highly restricted. Sialyl-Tn is an  O-glycans is considered as a common feature of breast and other carcinomas  that expresses STn and MUC1 by the transfection of MUC1 and ST6GalNAc I, the sialyltransferase responsible for the formation of STn, into 410.4 mammary tumour cell line. MUC1 glycopeptides, MUC1 glycoprotein and STn formulations were used as immunogens in an MUC1 transgenic model before challenging with the STn-expressing tumour cells. We found that Theratope (KLH-STn) vaccination induced tumour protection that was dependant on the expression of STn by the tumour cells and the presence of antibodies that could recognise a number of STn-carrying proteins.μMT alleles : polypeptide GalNAc-transferases as described  comprising three tandem repeats of MUC1. To obtain MUC1-pep-Tn, the same peptide was glycosylated O-glycosylation has been previously investigated and an average of 3.8 of the five potential sites of O-glycosylation of each tandem repeat was glycosylated  injection of 2.3 mg of cyclophosphamide. Theratope immunogen, provided by Biomira, was diluted in PBS and mixed with the enhanzyn adjuvant. Enhanzyn consists of Detox-B, which is an emulsion of monophosphoryl lipid A, mycobacterial cell wall and lecithin . Mice weμg of MUC1-pep-STn coupled to KLH or with 10 μg of MUC1-prot-STn mixed with Freund's adjuvant  in a total volume of 200 μl. Each animal received a total of four injections at two weekly intervals. Two weeks after the final injection the mice were challenged with 100 μl of 5 × 106 cells per ml E3-STn or E3 tumour cell suspended in PBS by s.c. injection into the flank.Three days before the first immunisation, mice were given an i.v. injection of 2.3 mg of cyclophosphamide. Mice were then injected s.c. with 10 Microtitre plates were coated with antigen overnight at 4°C with ovine submaxillary mucin , MUC1-pep-STn (50 ng per well), MUC1-pep-Tn (50 ng per well) or MUC1-pep-unglycosylated (50 ng per well) and the ELISA was performed as described previously /2, where a is the largest measure and b is the smallest in mm. Mice were killed when tumours reached 1–1.5 cm or if they ulcerated. Tumour-free survival was analysed using Kaplan–Meier model in SPSS software . Each animal was considered to be positive when a measurable tumour (13.5 mm3) was reported. Statistical significance was assessed using the Breslow test for which P<0.1 is considered significant.Tumour development was monitored three times per week by bi-dimensional measurement using vernier calipers. Tumour volumes in mmFrozen sections of human primary breast carcinomas, selected for their expression or lack of expression of STn, were fixed in ice-cold methanol/acetone (1 : 1) for 10 min. Sections were stained with mouse serum (diluted 1 : 400) or with the monoclonal antibody (mAb) to STn, TKH2 as described  were stained with the antibodies detailed in the Results followed by an biotinylated-anti-mouse IgG  diluted 1 : 200 and StreptABComplex/HRP (Dako) as previously described (M (pH 7.0), NaCl 150 mM, Triton X-100 2% (pH 7) for 4 h on ice and spun to remove large cell debris. Total cell lysate (6 mg protein) was then loaded into the column and the column was washed with 10 column volumes of PBS before elution with 0.1 M glycine at pH 2.5. The fractions were immediately neutralised and submitted to SDS–PAGE and western blotted with TKH2.TKH2 was coupled to CNBr-activated Sepharose 4 Fast Flow . E3-STn cells were lysed in Tris-HCl 50 m6 cells) were lysed in 50 mM Tris-HCl, 150 mM NaCl buffer containing 1% Triton X-100. Proteins (100 μg) or recombinant osteopontin  were loaded onto 4–12% gradient acrylamide gel, submitted to SDS–PAGE electrophoresis under reducing conditions and electro-transferred on nitrocellulose membranes in accordance with standard procedures.Cell pellets .M ammonium bicarbonate buffer  and extracted tryptic peptides were submitted to nano-HPLC-tandem mass spectrometry analysis as previously described . The gel was silver-stained and the bands of interest were cut and de-stained in a solution containing 1.6% sodium thiosulfate and 1% potassium ferrycyanate. After reduction and alkylation, proteins were submitted to in-gel trypsin digestion overnight at 37°C in 25 mmus musculus database  using the MS/MS ion search Mascot software . The search parameters were 1.5 Da tolerance for the parent ion mass  and 0.8 Da for the MS/MS fragment ions, one missed cleavage allowed, carbamidomethylcysteine as fixed modification and methionine oxidation as possible modification. Protein hits were ranking according to the protein scores derived from ions scores. Only the proteins with a significant score were considered (>35 with Swiss-Prot database).Fragment ion spectra were searched against the Swiss-Prot μl of A/G proteins agarose-beads (Roche) for 2 h and incubated with 2 μg of anti-OPN antibody for 3 h. A/G proteins beads were subsequently added and incubated overnight. Immunoprecipitated proteins were washed using TBS 0.05% Tween 20, the eluted from the bead using elution buffer (0.1 M glycine at pH 2.5) for two incubation of 15 min each. All procedures were carried out at 4°C using ice-cold reagents. The precipitates were then submitted to SDS–PAGE and western blotting as described above.Snap-frozen tumours were crushed using a Mikro-Dismembrator II . Tumour powders were dissolved in lysis buffer containing the Complete Mini anti-protease cocktail (Roche) and incubated for 2 h at 4°C. Lysates were homogenised and further incubated for 2 h. Total tumour lysates (0.5 mg of proteins) were pre-cleared using 50 To investigate the efficacy of STn to induce an immune response, STn conjugated to KLH in the form of Theratope was used to vaccinate both wild-type (WT) Balb/c mice and MUC1 transgenic mice on a Balb/c background. The MUC1 transgenic mice were selected as a model as in breast cancer MUC1 is the major mucin that carries STn. When Theratope was used in the clinical setting, patients were given low-dose cyclophosphamide with the aim of reducing their T-regulatory cells and so enhancing the immune response . Thus, bO-linked glycosylation sites carrying STn) but not with MUC1-pep-Tn  or MUC1-pep-unglycosylated (60mer)  . Further (60mer) , even thThe ability of anti-STn sera raised in MUC1 TG mice to react with various STn-positive proteins was analysed by western blotting. E3-STn and three STn-positive human breast cancer cell lines, MCF-7-STn, MDA-MB-231-STn . Identical results were obtained in the WT Balb/c mice (data not shown). To confirm the importance of the STn expression in the tumour protection, we challenged Theratope-vaccinated mice with E3 tumour cells not expressing the STn glycan. In this case, no delay in tumour growth was observed, with the Theratope-vaccinated mice developing tumours as quickly as the control group  or E3 that express human MUC1 without STn. roup see  and no dμMT KO mice, which are deficient in antibody production, with Theratope and tumour challenge with E3-STn cells as described above. Theratope increased tumour-free survival in WT Balb/c mice , demonstrating the essential role of antibodies in the tumour protection induced by Theratope immunisation.To ascertain the role of anti-STn antibodies in the tumour protection observed, we immunised Balb/c in vitro , MUC1-pep-STn coupled to KLH and MUC1-prot-STn, in a MUC1 transgenic mouse model to induce tumour protection. Despite all the immunised mice developing a strong humoral response we did not observe consistent tumour protection when the mice were vaccinated with MUC1-pep-STn or MUC1-prot-STn. However, further optimisation of the dose and delivery method may enhance the efficacy of the glycopeptide and glycoprotein used as immunogens. In contrast, immunisation with Theratope, thus making the immunogen independent of a specific peptide backbone, did result in a significant delay in tumour development. Furthermore, this effect was strictly dependant on STn being present in the tumour cells.μMT mice, which cannot produce immunoglobulins showed no delay in tumour growth after vaccination with Theratope, definitively proving that the tumour protection was dependant upon anti-STn antibodies. It can be noted that tumour appearance in μMT mice is delayed compared to WT mice. This phenomenon has previously been documented by The Theratope-immunised mice developed antibodies that recognised STn carried on MUC1, but also on several other proteins expressed by E3-STn cells or other STn-positive cell lines. Tumour challenge experiments performed in Balb/c O-glycoproteins carrying STn epitope, few of these proteins have been identified (Antibodies could delay tumour growth by antibody-dependent cellular cytotoxicity, inhibition of function or a combination of the two mechanisms, and indeed Theratope induced antibodies of the IgG2a subtype that mediate ADCC in mice . HoweverOsteopontin is expressed in most normal tissues during remodelling. It functions both as a cell attachment and chemotactic protein, mainly by interacting with integrins through an RGD domain, and also by interacting with CD44 (The results presented in this study demonstrate that STn glycans as present in Theratope can effectively produce tumour protection against STn-expressing cancer cells, which is strictly dependant on the induction of antibodies reactive with STn. The results also suggest that targeting STn carried on several proteins rather than only one is more likely to interfere with various mechanisms involved in tumour development. Moreover the targeting of multiple antigens may be advantageous in overcoming the problem of immunoediting that has been documented in a number of clinical studies  and alth


The vaso-active drug hydralazine causes a considerable increase in the cytotoxic effect of melphalan towards the KHT tumour in mice. The enhancement in response, measured as the concentration of melphalan required to achieve a given tumour response, is 3.0 and 2.35 when determined using the regrowth delay assay and the technique for determining surviving fraction in vitro following treatment in vivo respectively. In contrast, measurement of systemic toxicity shows that the addition of hydralazine only causes a small increase (ER = 1.15) in melphalan damage. This suggests that the drug combination may have some therapeutic benefit. The tumour specificity for the action of hydralazine is supported by the finding that binding of 3H-misonidazole is increased in tumours but not in other tissues when mice are treated with hydralazine. Increased binding of labelled misonidazole is associated with an increase in the level and duration of hypoxia, which will occur as a consequence of changes in tumour blood flow brought about by hydralazine. However, hypoxia per se is not responsible for the enhanced effect of melphalan, since the agent BW12C, which also induces substantial tumour hypoxia as a result of changing the O2 affinity of haemoglobin, has no effect on melphalan tumour cytotoxicity.


P < 0.05). The platelet counts were positively correlated with tumour size . Platelet counts were higher in the patients who had grade 3 microvessel density (P < 0.05). The mean basal platelet cGMP level in the patient group was significantly lower than it was in the control group (P < 0.05). Focal TSP immunoreactivity was detectable in 5 (20%) cases in the tumour cells, and in 9 (45%) cases in the stroma. We did not find any correlation between TSP-1 staining and angiogenesis, platelet counts, platelet activity, and the histological prognostic parameters. Our study confirms the essential role of platelets in tumour growth and angiogenesis. Decreased levels of cGMP in the patient group may cause platelet hyperreactivity. Although thrombospondin 1 may be upregulated in malignant breast tissue, this is not sufficient for tumour growth and dissemination according to our results.This study investigates angiogenesis and the expression of thrombospondin 1 in invasive ductal carcinoma of the breast and their possible relation to platelet counts and platelet activity. The study included 20 cases of invasive ductal carcinoma. Platelet activity was evaluated by determining thromboxane B2 and cyclic guanosine monophosphate (cGMP) levels by enzyme immunoassay (EIA).Thrombospondin (TSP) 1 and CD34 expression was studied immunohistochemically. Mean platelet count of the patient group was significantly greater than the mean platelet count of the control group ( Breast cancer is the most common carcinoma in women. Invasive ductal carcinoma not otherwise specified forms the largest group of invasive breast cancers . It compBreast cancer is a heterogeneous disease characterized by variable growth rates and metastatic potentials. Prognostic parameters which can be used to predict the behaviour of the tumours are sought. There are reports that observed that angiogenesis is an independent and significant prognostic marker for survival in early-stage breast carcinoma; several reports, however, have not been able to confirm the value of it .Thrombospondin (TSP) is a matricellular protein that functions as an angiogenesis inhibitor . StudiesPlatelets are the most abundant source of TSP. It is the major protein of platelet α granules and is expressed on the cell surface upon platelet activation . ExperimIn the present study, we evaluated angiogenesis and the expression of TSP-1 immunohistochemically in invasive ductal carcinoma of the breast and their possible relation to platelet counts and platelet activity in breast carcinoma patients. We have also analysed the effects of angiogenesis, TSP-1 expression, platelet counts, and platelet activation on prognostic histological parameters.We performed a prospective clinical and laboratory study. Procedures followed were approved by the Ankara University Human Ethics Committee, and they were in accordance with the Helsinki Declaration of 1975, revised 1983.The study group comprised 20 patients with primary operable invasive ductal carcinoma of the breast. All of the patients underwent modified radical mastectomy. The age range was 37–88 years, with a mean age of 56. They were non-smokers and were not prescribed acetylsalicylic acid in the two weeks prior to the operation.The control group for blood sample analysis included 14 healthy women with an age range of 34–76 years, with a mean age of 50 years. They were non-smokers and were not prescribed acetylsalicylic acid in the two weeks prior to sampling. From their histories of disease, it was evident that none had ever suffered from cancer or other chronic diseases.2PO4, 2.9 mM KCl, 1 mM MgCl2, 5 mM glucose, and 20 mM N-2-Hydroxyethylpiperazine-N′-2-Ethanesulphonic Acid (HEPES) (pH 7.4). The platelet count was adjusted to 300×109/L.Blood samples of patients were collected prior to the operation. Platelet-rich plasma (PRP) was obtained by centrifugation of citrated blood at 300 rpm for 15 min. Platelets were separated by centrifugation at 1200 rpm for 15 min and were washed twice before being resuspended in a HEPES-buffered Tyrode solution containing 138 mM NaCl, 0.36 mM NaHPlatelet activation was evaluated by determining thromboxane B2 (TxB2), the stable metabolite of thromboxane A2 (TxA2), and cyclic GMP cyclic guanosine monophosphate (cGMP) levels. Thrombin  in 0 8 U/ mL final concentration was added to the platelet suspension and was incubated for 15 min. The supernatant was prepared by centrifugation of thrombin-induced platelet suspension for 3 min at 10,000 rpm. TxB2 was evaluated by enzyme immunoassay (EIA) in the supernatants. The results were expressed as ng/mL ,13.For the determination of platelet cGMP content, PRP was incubated with sodium nitroprusside (SNP), a source of nitric oxide (NO), for 15 min, and ice-cold trisodium citrate was added. Platelets were pelleted by centrifugation, and 0.3 mL 96% ethanol was added. After evaporation of ethanol, 0.3 mL tris-ethylenediaminetetraacetic acid (EDTA) buffer was added. After 10 min, 0.1 mL of supernatant was tested by EIA . EIA kitSurgical resected specimens from 20 patients with operable breast carcinoma were used. Tumour size was reported as the largest diameter. Tumour size was graded as: 1+(≤2 cm), or 2+(>2 cm). Tumour samples were fixed in 10% neutral buffered formalin, embedded in paraffin, cut into sections of 3 µm thickness, and stained with haematoxylin-eosin. Histological diagnosis was invasive ductal carcinoma in all cases. Tumour grading, nodal status, and vascular invasion assessment were performed on haematoxylin-eosin-stained sections. Tumour grade was determined according to the criteria of the modified version of Bloom and Richardson's method . Status l-lysin-coated slides. The streptavidin-biotin-peroxidase method was performed using primary monoclonal antibodies against CD34 , TSP Ab-4 , oestrogen receptor (ER) , and progesterone receptor (PR) . Positive controls consisted of known positive samples of breast carcinoma for TSP Ab-4, ER, and PR. Normal identifiable vessels within the sections provided an internal control for CD34.The section with the deepest invasion was selected and marked for the immunohistochemical analysis. The representative blocks were sectioned and mounted on poly-Cytoplasmic staining in tumour cells and extracellular fibrillary staining in the stroma juxtaposed to tumour cells were defined positive. Semiquantitative analysis of the immunostaining was performed for each case. An estimate of the percentage of immunoreactive cells was determined using a score of 0–4 . The presence in the invasion front of a distinct TSP-positive cell zone composed of macrophages was scored into two categories: 1) No TSP-positive stromal macrophages or <10% TSP-positive cell zone composed of macrophages, 2) ≥10% TSP-positive cell zone composed of macrophages.2). Any endothelial cell or cell cluster positive for CD34 and clearly separate from an adjacent cluster was considered to be a single, countable microvessel  and progesterone receptor (PR) expression and considered as positive all cases displaying easily discernible nuclear staining in more than 5% of the tumour cells .U test. The correlations between the various parameters were determined by means of the Pearson test. A P-value of 0.05 or less was considered significant.Statistical analysis of the data was performed using Mann-Whitney P < 0.05)  (P < 0.05) (P < 0.05). There was no significant difference between TxB2 levels of the controls and the patients.There was no statistically significant difference between ages of control group and patient group. Although platelet counts were within normal ranges in both groups, the mean platelet count of the patient group was significantly greater than the mean platelet count of the control group ( < 0.05) . The pla < 0.01) . Platele < 0.05) . The meaP < 0.01). A TSP-positive cell zone composed of macrophages adjacent to tumour cells was observed in 7 (35%) of the tumours (The cellular distribution of TSP in tumour cells and macrophages was cytoplasmic with a granular texture. Fibrillary staining was observed in the stroma juxtaposed to tumour cells. TSP immunoreactivity ≤25% was detectable in 5 (20%) of the cases in the tumour cells, and in 9 (45%) of the cases in the stroma . There w tumours . No duct tumours .The Pearson test did not show the existence of any significant correlations between the tumour cell TSP staining, stromal TSP staining, TSP-positive cell zone composed of macrophages, MVD, and any of the conventional tumour features, including tumour size, tumour grade, vascular invasion, status of axillary lymph nodes, extranodal spread, ER immunostatus, and PR immunostatus. There was no correlation between TSP staining and MVD.Angiogenesis is the formation of new blood vessels from the endothelium of the existing vasculature and is essential for tumour growth and metastasis . It has Ultrastructural studies of experimental metastasis showed that circulating tumour cells are frequently surrounded by platelets . PlateleRecently the hypothesis that platelets are an important factor in angiogenesis has been proposed ,25. PlatTxA2 is the major vasoconstrictor and stimulator of platelet aggregation. There are inconclusive results about the role of TxA2 in tumour-induced platelet aggregation (TIPA) . ThrombiIntraplatelet cGMP acts to inhibit aggregation . It is ain situ TSP staining was confined to the basement membrane of the involved ducts. TSP could not be demonstrated in the basement membrane of normal breast epithelium  of the tumours in the tumour cells, and in 9 (45%) of the tumours in the stroma. There was a positive correlation between tumour cell TSP staining and stromal TSP staining. Intensity of staining ranged from weak to moderate. The tumours included in our study were grade 1 and grade 2. This may be the reason for weak to moderate staining of the tumours. Stronger staining may be observed if grade 3 tumours were included. A TSP-positive cell zone composed of macrophages was observed in 7 (%35) of the tumours. Tumour-infiltrating macrophages may be a major source of TSP-1 as well as tumour cells and tumour-associated stroma. We did not find any correlation between TSP-1 staining and angiogenesis, platelet counts, platelet activity, and the histological prognostic parameters we have studied. No ductal or stromal TSP staining was observed in adjacent normal breast tissue.The present study evaluated angiogenesis and expression of TSP-1 in invasive ductal carcinoma of the breast and their relation with platelet activity. The platelet counts were positively correlated with tumour size and were higher in the patients who had MVD grade 3. This confirms the essential role of platelets in tumour growth and angiogenesis. We found increased platelet counts and decreased cGMP levels in the patient group. Since cGMP level is accepted as an index of NO production, we thought that further studies are needed to explain the role NO-cGMP pathway in carcinogenesis and TIPA. Although TSP-1 may be upregulated in malignant breast tissue, this is not sufficient for tumour growth and dissemination according to our results.


The blocking of angiogenesis provides a novel therapeutic target to inhibit tumour spreading. In this study, we investigated the effect of linomide on angiogenesis induced in vivo by highly angiogenic breast carcinoma cells. The rabbit cornea was used to assess neovascular growth in the absence of a tumour mass. MCF-7 cells stably transfected with the cDNA encoding for vascular endothelial growth factor 121 (VEGF121) (V12 clone) were used to elicit a potent VEGF-dependent corneal angiogenesis. After tumour cell implant, albino rabbits received 100 mg kg(-1) day(-1) linomide for 5 consecutive days. Daily observation of neovascular progression indicated that linomide blocked angiogenesis. The antiangiogenic effect of linomide was apparent within 48 h from the beginning of the treatment and was both angiosuppressive and angiostatic. The block of neovascular growth lasted over 10 days from treatment suspension, and preformed vessels, which had regressed, remained dormant, suggesting the persistence of unfavourable conditions for capillary progression. Linomide (50-200 microg ml[-1]) was not cytotoxic in vitro on resting capillary endothelial cells but blocked endothelial cell replication induced by VEGF. Our data indicate that linomide can efficiently and persistently block VEGF-dependent angiogenesis in vivo in the absence of a growing tumour mass. These data suggest that linomide could be a chemopreventive drug in breast cancer patients and a valuable tool in clinical settings in which metastatic spreading occurs in the absence of a detectable tumour mass.


Results were expressed asa tumour/liver ratio. For comparison, treatments with Norcantharidin, Adriamycin and with hepatic artery ligation were studied. The mean tumour/liver ratio was 4.2+2.2 in untreated controls, but dropped significantly to 2.3+0.5 (p<0.05) with intra-arterial Norcantharidin (0.5 mg/kg) and to 2.3+0.7 (p<0.05) with intra-arterial Adriamycin (2.4 mg/kg), and to 2.2+0.7 (p<0.05) with hepatic artery ligation. However, with intravenous Adriamycin at 2.4 mg/kg, the mean tumour/liver ratio was reduced to only 3.5+2.0 and was not significantly different from untreated controls. It is concluded that intra-arterial Norcantharidin is as effective as intraarterial Adriamycin and hepatic artery ligation in suppressing tumour glucose oxidative metabolism. These results imply that Norcantharidin may have a role to play in the chemotherapy ofprimary livercancer.Norcantharidin is the demethylated form of Cantharidin, which is the active ingredient of the blister beetle, Mylabris, a long used Chinese traditional medicine. Though not well publicised outside China, Norcantharidin is known to possess significant anti-hepatoma activity, and is relatively free from side effects. In the present study, glucose oxidation in tumour and liver tissue slices harvested from hepatomabearing animals was quantified by measuring the radioactivity of


Growth of human tumours depends on the supply of oxygen and nutrients via the surrounding vasculature. Therefore tumour vasculature is an attractive target for anticancer therapy. Apart from angiogenesis inhibitors that compromise the formation of new blood vessels, a second class of specific anticancer drugs has been developed. These so-called vascular disrupting agents (VDAs) target the established tumour vasculature and cause an acute and pronounced shutdown of blood vessels resulting in an almost complete stop of blood flow, ultimately leading to selective tumour necrosis. As a number of VDAs are now being tested in clinical studies, we will discuss their mechanism of action and the results obtained in preclinical studies. Also data from clinical studies will be reviewed and some considerations with regard to the future development are given. It is a complex process in which vascular endothelial growth factor (VEGF) produced by tumour cells plays a predominant role  and positron emission tomography scans (PET) have been explored. This type of imaging can demonstrate changes in tumour perfusion and tumour viability  will be discussed.β-subunit of endothelial tubulin, resulting in depolymerisation of microtubules and disorganisation of actin and tubulin. Disruption of the endothelial cytoskeleton results in conformational changes leading to loss of blood flow. In addition to this, a recent study showed that the typical microtubule-destabilising agent combretastatin A4 phosphate (CA4P) also selectively disrupts the VE-cadherin/β-catenin complex interfering with cell–cell contact  (Combretastatin A4 phosphate is a water-soluble prodrug of combretastatin A4 (CA4). Following administration, CA4P is rapidly cleaved to CA4 and binds tubulin at or close to the colchicines-binding site . One of −2, the only drug-related toxicity was tumour pain in 35%. Tumour pain was not considered a dose-limiting toxicity (DLT) because it could be controlled by analgesics. Tumour viability and tumour blood flow were assessed by PET and DCE–MRI  and showed that CA4P was safe, well tolerated and lacking haematologic toxicity. In all studies MTDs of 50–60 mg m−2) 60 min after carboplatin (AUC 4–5). Dose-limiting toxicity was trombocytopenia  and asymptomatic decrease in left ventricular ejection fraction. Maximum-tolerated dose was set at 20 mg m−2. In all three studies ZD6126 was well tolerated and only showed mild side effects such as anaemia, nausea, vomiting and constipation. So far no objective tumour responses have been observed. Currently, ZD6126 is being explored in metastatic renal-cell carcinoma.In preclinical models this agent demonstrated significant antitumour activity. Stasis of blood flow was seen at doses 1/8–1/16 of the MTD and occurred specifically in tumour tissue . One patABT-751 is a sulphonamide molecule that can be given orally, and has shown significant antitumour activity in a variety of tumour models  , carboplatin (AUC 6) and DMXAA (1200 mg m−2). Preliminary data revealed no additional toxicity owing to the addition of DMXAA. Efficacy assessments are pending. In 78 patients with NSCLC, Two randomised phase-II studies combining DMXAA with conventional chemotherapeutics have recently been published. Vascular disrupting agents are a new class of antivascular anticancer agents that are currently undergoing clinical studies. At this moment, mainly (ongoing) phase-I studies have been presented, although some compounds have already entered phase-II testing either as single agent or in combination with chemotherapeutics. Therefore, the real (added) value in terms of patient benefit cannot be fully assessed yet.What distinguishes VDAs from other vascular targeting agents, how can we optimally assess their biological and clinical activity and how should these agents be taken forward?When assessing the toxicity pattern observed so far in the various clinical studies described, it is obvious that with regard to mechanism of action tumour specificity is most likely to be of critical importance. Vascular disrupting agents disrupt the established abnormal tumour vasculature by targeting the immature dysmorphic endothelial cells. As mentioned earlier tumour endothelium is more vulnerable to the activity of VDAs, and therefore in the end selective tumour vascular shutdown is likely to occur. However, based upon the pattern of side effects observed in clinical studies, normal vascular endothelium seems to be affected by VDAs as well. Cardiac ischaemia and cardiac arrhythmias as well as reversible neurologic complications  seem to underscore this issue and probably will remain dose limiting in future studies. Of critical importance therefore will be the assessment of biological activity at doses that can be administered safely. This will probably mean that in the design of early clinical studies the concept of looking for MTD will have to be replaced by the concept of looking for optimal biological dose, hereby assuming that the therapeutic window of these agents will permit us to do so. At this moment the probable optimal way to assess biologic or antivascular activity of VDAs is by repeated dynamic scanning, and therefore the role of DCE–MRI and PET analysis must be validated further. Vascular shutdown and decreased tumour blood flow as an indication of biological activity have meanwhile been demonstrated by DCE–MRI and/or PET analysis, whereas the occurrence of tumour pain following administration of several compounds also can be considered as an indication of biological and perhaps clinical activity. Whether biological activity will result in tumour size reduction and meaningful patient benefit has to be evaluated further in phase II and randomised phase-III studies. The assessment of cardiac and neurological complications that can occur in these studies necessitates optimal communication between oncologists and other specialists, and this will also be important when patients willing to be enrolled in studies are being screened . This isWhen looking at the observed biological effects induced by VDAs, the induction of central tumour necrosis whereas leaving a viable rim at the periphery seems to be a consistent finding. Probably, this means that there is a rationale to combine VDAs with other treatment strategies. Many theoretical combinations can be thought of, and combining VDAs with conventional cytotoxic treatment is already being pursued to a rather large extent. Apart from this, the addition of an angiogenesis inhibiting agent following VDA administration conceivably could induce ‘synergistic’ antiangiogenic activity leading to a complete growth inhibition and a subsequent state of dormancy of the ‘centrally killed’ tumour mass. This observation has meanwhile indeed been made in preclinical models . In addiExploring a combination of VDAs and epidermal growth factor receptor (EGFR) inhibitors theoretically could also be an interesting approach; here one could speculate that tumour cells in the viable rim will become apoptotic and die when being deprived of their growth-stimulating factors such as EGF.Effectiveness of combination therapies often depends on the sequence of administration. First increasing the vascular permeability (within minutes) allowing accumulation of a cytotoxic agent in the tumour and then inducing a shutdown of blood flow (within hours) could probably kill large amounts of tumour cells. However, as certain chemotherapeutics have their own vascular side effects , the alternative sequence of first administering the VDA followed by the administration of a cytotoxic drug could also be considered. Irrespective of the sequence administered, these combination therapies should be carefully chosen and closely monitored.From a theoretical perspective, radiation therapy probably must precede VDA administration because optimal blood flow and oxygen radical formation might be necessary for an optimal synergistic effect. Preclinical models have meanwhile shown such a synergistic effect. Finally, and considered as a very specific example, in tumours of the extremities, one could think of isolated limb perfusion with VDAs after clamping; Biologically active concentrations of VDAs could probably be achieved with only limited systemic exposure, thus leading to optimal and selective tumour cell killing while preserving the heart, central nervous system and other potential organs at risk. Monitoring antivascular effects with either angiography, DCE–MRI or PET would be challenging.In conclusion, VDAs are a new and promising class of targeted anticancer agents. Although their safety currently is the major focus of research, results that will show anticancer activity are likely to follow soon.Their unique mechanism of action merits thorough and extensive exploration, both as single agent as well as in combination with other treatment modalities. If these studies are performed adequately, with a close observation of toxicity, it is to be expected that in the years to come a clear picture of their role in anticancer treatment can be established.


DT-diaphorase is a two-electron-reducing enzyme that is an important activator of bioreductive anti-tumour agents, such as mitomycin C (MMC) and EO9, and is inducible by many compounds, including 1,2-dithiole-3-thiones (D3Ts). We showed previously that D3T selectively increased DT-diaphorase activity in mouse lymphoma cells compared with normal mouse marrow cells, and also increased MMC or EO9 cytotoxic activity in the lymphoma cells with only minor effects in the marrow cells. In this study, we found that D3T significantly increased DT-diaphorase activity in 28 of 38 human tumour cell lines representing ten tissue types with no obvious relationships between the tumour type, or the base level of DT-diaphorase activity, and the ability of D3T to increase the enzyme activity. Induction of DT-diaphorase activity in human tumour cell lines by 12 D3T analogues varied markedly with the D3T structure. D3T also increased DT-diaphorase activity in normal human bone marrow and kidney cells but the increases were small in these cells. In addition, D3T increased the level of enzyme activity in normal human lung cells. Pretreatment of human tumour cells with D3T analogues significantly increased the cytotoxic activity of MMC or EO9 in these cells, and the level of enhancement of anti-tumour activity paralleled the level of DT-diaphorase induction. In contrast, D3T did not effect the toxicity of EO9 in normal kidney cells. These results demonstrate that D3T analogues can increase DT-diaphorase activity in a wide variety of human tumour cells and that this effect can enhance the anti-tumour activity of the bioreductive agents MMC and EO9.


Spleen cells from mice immunized against a methylcholanthrene induced rhabdomyosarcoma inhibited tumour cell colony formation in vitro and prevented tumour development in vivo in an adoptive transfer test. Treatment of the immune spleen cells with anti-mouse immunoglobulin serum or passage through a nylon wool column, both of which reduced the percentage of immunoglobulin bearing cells in the population to less than 3-4%, did not alter their anti-tumour effects. In contrast, treatment of the spleen cells with anti-BAomicron serum abolished their anti-tumour effects both in vitro and in vivo. These results indicate that T cells are the mediators of tumour immunity in this chemically induced tumour system.


Previous studies have shown 5,6-MeXAA to be curative in 80% of mice bearing colon 38 tumours and 12 times more dose potent than FAA. This investigation has demonstrated that a murine colon tumour cell line (MAC15A) is approximately 60 times more sensitive to 5,6-MeXAA than to FAA, although these differences were not seen in three human cell lines tested. 5,6-MeXAA caused significant blood flow shutdown and haemorrhagic necrosis in subcutaneous MAC15A tumours in syngeneic and nude hosts, but measurable changes in tumour volume were seen only in syngeneic hosts. 5,6-MeXAA was inactive against intraperitoneal MAC15A but produced significant anti-tumour effects against the same cell line inoculated via an intravenous route. FAA has been shown previously to be inactive in this model. Interestingly, the effects against lung colonies were not accompanied by obvious necrotic changes, suggesting that they may be the result of increased direct cytotoxicity rather than an indirect host mechanism. Further studies to investigate the effects against systemic tumour deposits are under way.


In the absence of gross deformity of the nipple, such as its retraction or Paget's disease, histological examination of this area is often neglected, or at best confined to a cursory look at a single sagittal section. The inadequacy of this approach is illustrated by this study of 33 consecutive cases of carcinoma of the breast treated with mastectomy. Multiple transverse sections showed tumour in 19 nipples (58%) involving one or more levels. Of these, 17 showed non-invasive tumour, either ductal or lobular type. Invasive tumour was seen in only two nipples, one of which was metastatic extension from the underlying breast tumour. Paget's cells were seen in two cases. The most significant finding was the eccentric location of tumour in 14 nipples. A single central sagittal section would have detected only five cases involving the centrally situated duct. An inexplicable finding was a preponderance of right nipple with tumour. No statistically significant correlation between nipple involvement and the size, location, multicentricity, type of tumour in the breast and metastases in axillary lymph nodes could be found. It became evident from this study that malignant changes in the nipple occur more commonly than is realised, and that it is also one of the sites of multicentric origin of the tumour. This factor will have to be taken into account in planning conservative therapeutic programmes.


The 32P uptake in a mammary tumour induced by DMBA and in the Walker 256 carcinosarcoma was measured by external GM -tubes. The uptake was significantly higher than in the skin. During exposure to a synchronized light regime a circadian variation was present in the 32P uptake of the hormone-dependent DMBA-induced tumour. The maximal 32P uptake was in the dark period, in which the highest temperature in the tumour has also been found . In the hormone-independent Walker 256 carcinosarcoma there was no periodicity in 32P uptake. No variation in 32P uptake was registered in the skin of normal controls or in tumour-bearing rats.


The small fragments of tumour used in this technique were also effectively stained with fluorescent probe-conjugated antibodies, allowing characterisation of the vessels based on surface marker phenotype. The vessels within tumour tissue were much more irregular and tortuous compared to those within surrounding normal muscle. B16 tumours stably transfected with the genes for IL-12 were used to assess the effects of this cytokine on tumour growth and vessel formation. The IL-12-expressing tumours grew more slowly and had much smaller blood vessels than the large, webbed vessels characteristic of the parental tumours, effects that were dependent on interferon gamma (IFN-γ). Vessels in the parental tumours were found to express VEGFR-3, the receptor for VEGF-C and VEGF-D. Expression of this receptor by the endothelial cells of the blood vessels was lost in the cytokine expressing tumours, thus suggesting a mechanism for the antiangiogenic effects of IL-12. The combination of the whole mount technique and the GFP transgenic mice provides a powerful method for visualising tumour vasculature and characterising the effects of agents such as cytokines.New blood vessel formation within tumours is a critical feature for tumour growth. A major limitation in understanding this complex process has been the inability to visualise and analyse vessel formation. Here, we report on the development of a whole-tissue mount technique that allows visualisation of vessel structure. Mice expressing green fluorescent protein (GFP) made it possible to easily see GFP Angiogenesis is a multistep process that is limited and carefully regulated in normal adult tissue, but in tumours this regulation is disrupted and the process remains ‘switched on’ . Ample etm1TsIfng (IFN-γ deficient) mice were purchased from the Jackson Laboratory . Green fluorescent protein-transgenic mice (C57BL/6-H-2KbP-GFP) were generated as previously described  in which KpnI/XbaI sites were created by removing the EGFP gene. The final construct contained both IL-12 genes and a neo-selectable marker.The expression vector pmIL-12-neo.1 was made by excising a 4405-bp fragment from pWRG 3169 (a kind gift from Dr Hua Yu (b) spontaneously arising melanoma, was obtained from the ATCC (CRL 6322) and maintained in MAT/P medium (USA patent #4.816.401) supplemented with 100 U ml−1 penicillin, 100 mg ml−1 streptomycin and 2% fetal calf serum. The B16 IL-12-expressing line (B16/IL-12) was constructed using the pmIL-12-neo.1 plasmid as described above. B16-F0 cells were transfected using Lipofectin , according to the manufacturer's directions. At 24 h after transfection, selection of transfectants was initiated with 400 μg ml−1 G418 ; 10–14 days later, cells were cloned at limiting dilution. Clones were expanded and tested for IL-12 (p70) production by ELISA. This assay was specific for IL-12 p70 by virtue of the use of a rat anti-mouse IL-12 (p70) capture antibody , which reacts with the p70 heterodimer but does not react with the p40 subunit. Bound IL-12 (p70) was then detected with a biotin-conjugated anti-mouse IL-12 (p70/p40) antibody . All washes were carried out in the absence of azide, and a recombinant IL-12 (p70) standard (BD Pharmingen) at 4000 pg ml−1 was run in each assay.The B16-F0 cell line, a C57BL/6   were injected intramuscularly into the thighs of C57BL/6 or C57BL/6-H-2KbP-GFP mice. Mean thigh diameter was determined as described previously (Tumour cells in 100 μm). To ensure uniform thickness we subsequently constructed a frame mounted to a glass slide, which placed uniform pressure on the entire perimeter of the coverslip (using screws machined to stop at 100 μm) so that each fragment would be 100 μm in depth. Owing to the heterogeneity inherent both within and between tumours, several pieces (six to nine) from different areas were examined from each tumour and several tumours (three to six) were used in each study.Whole mount histology was performed by excising small pieces (∼2 mm × 3 mm × 1 mm) from the tumour and placing it on a glass slide with two drops of PBA  . A coverslip was placed on top of the piece of tumour, gently pressed down and excess PBA removed by blotting with absorbent tissue. This tumour fragment was then viewed via fluorescence microscopy. Analysis of these fragments by confocal microscopy showed them to be of fairly uniform thickness  was added at 1 : 400 in 200 μl PBA and incubated with shaking at 4°C for 1–1.5 h, followed by washing as described above. After the final wash, samples were removed from the tubes using 1000 μl pipette tips with tip cutoff, and placed on slides as described above. To control for possible nonspecific binding of the antibodies, additional samples were stained with either a directly conjugated antibody of the same isotype as the primary antibody, or unconjugated antibody of the same isotype followed by streptavidin–biotin or streptavidin–biotin alone. In all cases, background staining was extremely low or nonexistent.Samples to be stained with antibodies were placed in 6 ml polypropylene tubes, and the samples blocked using Fc Block  at 10 μm cryostat sections mounted on poly-L-lysine coated slides and stored at −80°C. Staining was performed on unfixed sections, blocked with Fc block and stained as for the whole mount technique except that 100 μl of antibodies, diluted in PBA to the predetermined concentration were placed on the sections and the slides incubated in a humid box.Conventional immunohistochemistry was performed using 4 The percent vessel area was determined on antibody-stained whole mounts prepared as described above. Each tumour was stained with phycoerythrin (PE)-conjugated anti-CD31 or anti-panendothelial cell (PanEC) , and 30–35 digital images (× 10 objective) were obtained per tumour. Images were analysed using Image Pro Software by colour segmenting each image using a predefined colour range to form a binary image of the tumour blood vessels. From these images, percent vessel area was then computed for each image and averaged to obtain percent vascular area. Tumour vessel diameters were determined on whole mounts stained in the same manner. For these determinations 25–30 digital images (× 10 objective) were obtained per tumour. In each image, vessels were manually selected and diameters of all vessels were determined, using Image Pro Manual Measurement techniques, and exported to Microsoft Excel for statistical analysis. More than 500 (524–1323) vessels were measured for each tumour.+ structures, thus confirming that they were indeed blood vessels. Fragments of the brain tissue were incubated with a PE-conjugated anti-class I antibody, which also stained the blood vessels quite brightly, whereas the surrounding brain tissue was largely unstained  encompassing an area of approximately 3.2 × 4.2 mm were prepared from pieces of size matched B16 and B16/IL-12 tumours . In the B16/IL-12 tumours, GFP+ blood vessels were also clearly visible, but these were completely unstained with the anti-VEGFR-3 antibody (bottom row of images), even though other cell types are seen to be VEGFR-3 positive. To verify this finding, additional tumours were examined by conventional immunohistochemistry  and its corresponding receptors (VEGFR). Antibodies to VEGFR-1, VEGFR-2 and VEGFR-3 were used to stain vessels from the parental and IL-12-expressing tumours. Both VEGFR-1 and VEGFR-2 were found on vessels from both types of tumours and thus were unlikely to account for the differences in vasculature between the two types of tumour (data not shown). In contrast, VEGFR-3 expression, which was clearly present on the vessels from the parental tumours, was highly downregulated on the vessels from the IL-12-expressing tumours. This can be clearly seen in the images in Figure 7Ahemistry . For theμm or less) and since many of the vessels are cut as cross-sections, this means that only two to three cells are being observed, resulting in limited fluorescence intensity. Even with frozen sections, it is typical to use some fixation method, which tends to affect the GFP fluorescence intensity adversely. In contrast, the whole mount method uses tissue fragments, making it possible to view large segments  of essentially intact vessels. The tissue is neither fixed nor frozen, so GFP expression is maintained at a high level within the viable cells. Compared to conventional immunohistochemistry, this method is simpler, faster and provides a more comprehensive view of the vascular configuration within the tumour tissue.This study describes and validates a new whole mount method for visualising and characterising the blood vessels within experimental mouse tumours. The use of transgenic mice expressing GFP under the control of the class I promotor greatly assisted verification of the technique. In initial studies, we attempted to use conventional immunocytochemistry to visualise blood vessels, but encountered problems in detection of the GFP fluorescence (data not shown), as has been previously described . Several+ vessels were colocalized. The ability to stain with antibodies greatly expands the power of the whole mount technique in that it enables one to use the large number of fluorochrome-conjugated reagents that have been developed for flow cytometry. Thus, this technique combines many of the best features of flow cytometry and immunohistochemistry in a method that is simple to perform.Since the implanted tumours are GFP negative, they provide a blank background on which the bright GFP fluorescent blood vessels of host origin are easily seen. To further characterise the blood vessels, we used fluorochrome-conjugated antibodies specific for surface markers present on the endothelial cells. Despite the relatively large size of these antibody molecules, they were found to diffuse throughout the tissue. All of the antibodies used in these experiments were of the IgG class, which is known to be present in tissues and may have been selected over evolutionary time to be able to penetrate many tissues and organs easily. Early experiments established the effectiveness of the staining by verifying that PE-anti-CD31-stained vessels and GFPWe have used this technique to determine how IL-12 alters the angiogenic properties of tumour cells. Our results indicate that IL-12 inhibits angiogenesis in B16 mouse tumours by downregulating the expression of VEGFR-3. By using B16 tumour cells transfected with the genes for IL-12, we were able to grow tumours in which the cytokine was produced and secreted locally within the tumour microenvironment. This ensured high local concentrations of the cytokine while limiting side effects evident when this cytokine is delivered systemically (in vivo (data not shown). Initially, it was thought that VEGFR-2 was expressed on both blood vascular and lymphatic endothelial cells, whereas VEGFR-3 expression was limited to lymphatic vessels and fenestrated endothelium , and the vessel morphology was similar  to that of the vessels in the parental tumour and the vessels expressed the VEGFR-3 (data not shown). In some tumour models, the IL-12 effects have been shown to require tumour cells that are responsive to IFN-γ  cells can affect the function of endothelial cells (The IL-12-mediated effects we observed in this model tumour system appear to be mediated primarily via IFN-+ transgenic mice, we have been able to visualise the vasculature within B16 tumours and observe the effects of locally produced IL-12 on the tumour vessels. This technique combined with immunohistochemistry has allowed us to determine that the antiangiogenic effect of IL-12, at least in this tumour model is related to the marked downregulation of VEGFR-3 expression on the endothelial cells of the tumour vasculature. These results emphasise the importance of these growth factor receptors in the angiogenic process and suggest new possibilities for tumour control.By using whole mounts of viable tumour tissue growing in GFP


C. parvum on intrastrain transplants of a mammary carcinoma in A/HeJ mice has been confirmed, and it has been shown further that C. parvum inhibits the growth of transplants of sarcomata induced with methylcholanthrene both in this strain (members of which lack the fifth component of complement) and in CBA mice (which are not complement deficient). In experiments with the mammary carcinoma, 2 injections of C. parvum on days + 3 and + 9 were more effective than a single injection on day + 3; injections on days + 3 and + 6, or + 3 and + 12, appeared to be marginally less effective than on days + 3 and + 9, but the difference was not statistically significant.The inhibitory effect of an i.v. or i.p. injection of C. parvum, the tumour cells were pre-incubated with anti-tumour globulin (ATG) in the absence of complement prior to inoculation, and the effect of combining these procedures was much greater than that of either alone. Pre-incubation with ATG had a similar but less marked effect on the development of the mammary carcinoma but had no effect on the A/HeJ sarcoma. Injection (i.v.) of ATG did not inhibit the growth of any of the tumours in these experiments and possible reasons for this are discussed.Development of the CBA sarcoma was inhibited to about the same extent if, instead of treating the mouse with


Niosomes are multilamellar vesicles formed from nonionic surfactants of the alkyl or dialkyl polyglycerol ether class and cholesterol. Adriamycin has been trapped within vesicles prepared from a monoalkyl triglycerol ether and its activity compared with adriamycin solution in human lung tumour cells grown in monolayer and spheroid culture and in tumour xenografted nude mice. The activity of the encapsulated adriamycin in vitro is maintained with similar clonogenic survival curves following treatment of monolayers and identical growth delays following spheroid exposure. The pharmacokinetics of adriamycin are altered in vivo in human lung tumour-bearing nude mice, when it is administered in niosomal form. There is prolonged release of drug from the plasma compartment with significantly lower peak levels; lower peak cardiac adriamycin concentrations with a shorter tissue half-life and decreased cardiac AUC and a greater degree of hepatic metabolism to inactive 7-deoxyaglycones. The tumour peak drug level and AUC was similar irrespective of the mode of administration of adriamycin. The growth delay (i.e. the time taken for the tumour volume to double) was significantly longer for adriamycin (15 days) and niosomal adriamycin (11 days) than for control (5.8 days). It is possible that the therapeutic ratio of adriamycin could be enhanced by administration in niosomal form.


P<0.01). On univariate analysis, both C-reactive protein concentrations (P<0.001) and percentage tumour volume of CD4+ (P<0.05) T-lymphocytes were associated with cancer-specific survival. The results of the present study show that low tumour CD4+ T-lymphocyte infiltration is associated with elevated C-reactive protein concentrations and both predict poor cancer-specific survival.There is increasing evidence that both local and systemic inflammatory responses play an important role in the progression of a variety of common solid tumours. The aim of the present study was to examine the relationship between tumour T-lymphocyte subset infiltration, the systemic inflammatory response and cancer-specific survival in patients with colorectal cancer. In all, 147 patients undergoing potentially curative resection for colorectal cancer were studied. Circulating concentrations of C-reactive protein were measured prior to surgery. CD4+ and CD8+ T-lymphocyte infiltration of the tumour was assessed using immunohistochemistry and a point counting technique. When patients were grouped according to the percentage tumour volume of CD4+ T-lymphocytes, there was no difference in terms of age, sex, tumour site, stage and tumour characteristics. However, there was an inverse relationship between percentage tumour CD4+ T-lymphocytes and C-reactive protein ( It has long been recognised that disease progression in cancer patients is not solely determined by the characteristics of the tumour but also by the host response. Indeed, there is increasing evidence that both local and systemic inflammatory responses play an important role in the progression of a variety of common solid tumours .In patients with colorectal cancer, there is good evidence that, on simple staining of tumour sections, the presence of a pronounced lymphocytic infiltration within the tumour is associated with improved survival . The limit of detection of the assay is a CRP concentration lower than 5 mg lThe study was approved by the local ethics committee.μm) were cut and mounted on slides coated with aminopropyltriethoxysilane. Sections were then immunostained using the peroxidase-based Envision  technique as described previously . The observers (Canna and McArdle) were blinded to the clinical outcome of the patient.χ2) and the Kruskal–Wallis test for analysis of variance. For the purpose of analysis, T-lymphocyte subsets were grouped by tertiles as described previously  who underwent potentially curative resection for colorectal cancer are shown in The baseline clinicopathological characteristics of the patients . There was a positive relationship between percentage tumour CD4+ and CD8+ T-lymphocytes .Patients grouped according to tertiles of the percentage tumour volume of CD4+ T-lymphocytes are shown in P<0.001), sex (P=0.052), Dukes' stage (P<0.001) and venous invasion (P=0.002) were associated with poorer cancer-specific survival. A decreased percentage tumour volume of CD4+ T-lymphocytes .The minimum follow-up was 30 months; the median follow-up of the survivors was 62 months. During the course of the study, 55 patients died, 39 patients of their cancer and 16 of intercurrent disease. On univariate analysis, increased age , stage  and C-reactive protein  retained independent significance.On multivariate analysis, only age  was superior to tumour T-lymphocytic infiltration  in predicting cancer specific survival. One possible explanation is that C-reactive protein can be measured with greater accuracy and precision than tumour T-lymphocytic infiltration. Alternatively, the systemic inflammatory response may be more important in determining survival in these patients.The relationship between tumour CD4+ T-lymphocytic infiltration and cancer-specific survival is the opposite of that previously reported for both renal and prostate cancer  and thatIt is of interest therefore that In colorectal cancer, it has been reported that interleukin-6 concentrations increase with tumour stage and correlate with CEA concentrations. This might therefore suggest that interleukin-6 is produced by the tumour cells (In the present study, we attempted to assess interleukin-6 within the tumour using different methods of antigen retrieval and staining and the use of negative and positive controls. However, we were unable to reliably identify regions of IL-6 expression in the colorectal tumours due to deep background staining, which precluded accurate scoring of IL-6-positive cells in the tumour tissue.In summary, the results of the present study show that, in patients undergoing curative resection for colorectal cancer, low tumour CD4+ T-lymphocyte infiltration is associated with elevated C-reactive protein concentrations. Furthermore, both a low tumour CD4+ T-lymphocyte infiltration and an elevated C-reactive protein predict poor cancer-specific survival.


Electrochemotherapy is a combined use of certain chemotherapeutic drugs and electric pulses applied to the treated tumour nodule. Local application of electric pulses to the tumour increases drug delivery into cells, specifically at the site of electric pulse application. Drug uptake by delivery of electric pulses is increased for only those chemotherapeutic drugs whose transport through the plasma membrane is impeded. Among many drugs that have been tested so far, bleomycin and cisplatin found their way from preclinical testing to clinical use. Clinical data collected within a number of clinical studies indicate that approximately 80% of the treated cutaneous and subcutaneous tumour nodules of different malignancies are in an objective response, from these, approximately 70% in complete response after a single application of electrochemotherapy. Usually only one treatment is needed, however, electrochemotherapy can be repeated several times every few weeks with equal effectiveness each time. The treatment results in an effective eradication of the treated nodules, with a good cosmetic effect without tissue scarring. Detailed information about the treatment is published in Standard Operating Procedures (SOP) 2.The treatment procedure for electrochemotherapy consists of local or systemic drug injection followed by delivery of electric pulses applied to the tumour Tumour type: basal cell carcinoma, and cutaneous metastases of malignant melanoma, breast carcinoma, hypernephroma, Kaposi's sarcoma and others.Tumour location: accessible cutaneous and subcutaneous tumour nodules on all parts of the body.Tumour size can be from 3 mm to 3-5 cm in diameter.Number of nodules; from 1 to up to 100.Local or general anaesthesia, depending on the number of the treated nodules and the location.2. Injected dose for intratumoural application of bleomycin is 250-1000 IU/cm3 of tumour tissue and for cisplatin 0.5 -1 mg/cm3 of tumour tissue Drug choice: cisplatin or bleomycin. Cisplatin and bleomycin can be injected intratumorally, bleomycin also intravenously in a dose of 15000 IU/mTime interval between the drug administration and application of electric pulses; after intratumoral injection of the drug immediately, after intravenous administration after 8 min. up to 28 min.Electric pulse generator for electrochemotherapy should provide square wave electric pulses of amplitude 1000 V or more, repetition frequency from 1 Hz to 5 kHz.Electric pulse parameters: 8 pulses of e.g. 1000 V at 8 mm distance between two plate electrodes, frequency 1 Hz or 5 kHz, duration of the pulse 100 µs.If the whole tumour can not be encompassed between the electrodes, application of electric pulses should be repeated that many times so as to cover the whole tumour area, including safety margins.Electrode type: plate electrodes are used for small, superficial tumour nodules, needle electrodes are for treatment of deeper seeded nodules under the skin.Usually single treatment is effective for tumour nodule eradication; however electrochemotherapy can be repeated in 4 weeks intervals on the remaining tumour tissue or other tumour nodules.Due to lack of systemic toxicity - because very low doses of bleomycin or cisplatin are needed - patients can be treated on an out-patient basis, and can leave the hospital soon after the treatment.3,4. Thereafter electrochemotherapy was demonstrated to be effective also for cisplatin 5. Extensive preclinical data were collected on in vitro and in vivo tumour models in the following years. Treatment effectiveness was determined in relation to drug dosage, route of its administration, timing of drug injection and application of electric pulses, intensity of the electric field, coverage with sufficiently high electric field (E), and appropriate selection of electrodes and positioning with respect to the tumour 1,6,7,8. Furthermore, mechanisms underlying the effectiveness of electrochemotherapy were elaborated, demonstrating that, besides direct effect of electrochemotherapy to tumour cells, vascular disrupting effect and immune response are involved 1,6,9. All of these collected data enabled translation of electrochemotherapy into the clinics.Increase in cytotoxicity of bleomycin by exposure of cells to electroporative electric pulses was first described by Okino M and Mir LM 10. Later on, its effectiveness was demonstrated on other tumour types, such as basal cell carcinoma of the skin, cutaneous metastases of melanoma, mammary tumours, hypernephroma and Kaposi's sarcoma. There are several reports evaluating collectively all clinical data published on electrochemotherapy with bleomycin and cisplatin 11-15. Overall, the response rate of the treated tumours was approximately 80% objective responses and approximately 70% complete responses 15,16. The effectiveness can be even higher by repetitive treatment 17.The first clinical trials demonstrated effectiveness of electrochemotherapy on head and neck and melanoma tumour nodules All of these clinical data have enabled electrochemotherapy to be adopted in some European countries as standard treatment, with palliative intent, on various tumours. The future of this treatment is to introduce electrochemotherapy in treatment of internal tumours and metastases, and in combined treatment, either with gene therapy or radiation therapy. These attempts are already under way.


Monoclonal antibodies to epithelial-cell antigenic determinants, labelled with 123I and 125I, were administered parenterally to immunodeficient mice bearing human tumours derived from a human cancer cell line. Anterior, posterior and lateral radioscans of the body were taken with a gamma scintillation camera at various times from immediately to 65 days after injection. Visual displays of the images were processed by standard computer techniques. The model used a human colon-cancer cell line, HT29, and the monoclonal antibody, AUA1, which is specific to an epithelial proliferating antigen. Tumour detection was achieved in all the mice. The smallest tumour detectable appeared to be about 1 mm in diameter. The degree of antibody uptake in a tumour depended on its size and the blood supply of its surrounding tissues We believe that the technology and skills are now available for accurate radioimmunodetection of cancer in man.