Two mouse monoclonal antibodies (MoAbs), TP-1 and TP-3, previously shown in immunohistochemical studies to react with osteosarcomas, were labelled with 125I or 131I and evaluated for their ability to localise to human osteogenic sarcoma xenografts after intravenous injection. The radiolabelled TP-1 and TP-3 MoAbs had immunoreactive fractions of 70% and 67%, respectively, and bound to target cells with binding constants of 8.5 X 10(8) M-1 and 4.0 X 10(9) M-1, respectively. After injection of labelled TP-3 IgG, approximately 16% of the dose X g-1 tissue was found in the tumour after 24 hours. Maximum tumour/blood radioactivity ratios of 6-7 were achieved 3-4 days after antibody injection, while the ratios for the normal tissues were less than 1. The tumours could be clearly visualised by whole-body gamma scintigraphy without the need for subtraction techniques. The TP-1 IgG accumulated to a large extent also in the spleen. Hence, with this antibody the tumour was less well delineated from the adjacent normal tissues. However, the F(ab')2 fragments, derived from the TP-1 IgG, gave tumour/blood ratios up to approximately 40 after 3-4 days and yielded sharp gamma scintigrams of the tumour. Specificity of the antibody localisation was indicated by the lack of accumulation in a contralateral melanoma xenograft and the failure of 2 isotype-matched irrelevant MoAbs to localise to the sarcomas. With the F(ab')2 fragments satisfactory images could be obtained already after 16 hours. The results suggest that this preparation may be useful in clinical radioimmunodetection of osteogenic sarcomas.


Reactive oxygen species including hydroxyl radicals can cause oxidative stress and mutations. Inhaled particulate matter can trigger formation of hydroxyl radicals, which have been implicated as one of the causes of particulate-induced lung disease. The extreme reactivity of hydroxyl radicals presents challenges to their detection and quantification. Here, three fluorescein derivatives  and two radical species, proxyl fluorescamine and tempo-9-ac have been compared for their usefulness to measure hydroxyl radicals generated in two different systems: a solution containing ferrous iron and a suspension of pyrite particles.APF, amplex ultrared, and DCFH react similarly to the presence of hydroxyl radicals. Proxyl fluorescamine and tempo-9-ac do not react with hydroxyl radicals directly, which reduces their sensitivity. Since both DCFH and amplex ultrared will react with reactive oxygen species other than hydroxyl radicals and another highly reactive species, peroxynitite, they lack specificity.The most useful probe evaluated here for hydroxyl radicals formed from cell-free particle suspensions is APF due to its sensitivity and selectivity. Hydroxyl radicals (•OH) have been implicated in genotoxicity  . In addiemically , its useemically ,20,21. Ad breaks .•OH. Three of the five probes evaluated will react directly with •OH, while the other two require reaction of •OH with another molecule that can directly react with the probes.For this study, our objective was to evaluate several hydroxyl radical detection techniques that could overcome some of the challenges described in the preceding paragraph. Several relatively new compounds have been developed for ROS detection using fluorescence. Some of them will detect many ROS and others have been designed to react only with highly reactive oxygen species such as p-aminophenyl) fluorescein (APF), 10-acetyl-3,7-dihydroxyphenoxazine (amplex ultrared), 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), 5-(2-carboxyphenyl)-5-hydroxy-1-methyl)-3-phenyl-2-pyrrolin-4-one, potassium salt (proxyl fluorescamine), and 4-((9-acridinecarbonyl)amino)-2,2,6,6-tetramethylpiperidin-1-oxyl (tempo-9-ac).Five probes were evaluated: 3'- . Under these conditions, the hydroxyl radicals react with the DMSO to form methyl radicals, which then react with proxyl fluorescamine to yield the fluorescent hydroxylamine derivative of fluorescamine. Proxyl fluorescamine has been used to detect ROS generated by suspended diesel exhaust particles -2,2,6,6-tetramethylpiperidine (acridine-piperidine) ; however, predicting the concentration of hydroxyl radicals that are produced in a Fenton reaction is exceedingly difficult. Even if all of the H2O2 were to stoichiometrically form •OH, any remaining Fe(II) may form additional H2O2 through other reactions (eqs 1 to 2). Instead of adding Fenton reagents to the probes, a known concentration of hydrogen peroxide was added in the presence of horseradish peroxidase (HRP). Peroxidases catalyze the oxidation of an organic substrate by facilitating electron transfer with hydrogen peroxide. Even in the presence of a peroxidase, however, proxyl fluorescamine and tempo-9-ac will not react efficiently with H2O2. As indicated above, a carbon radical, derived from exogenously added dimethyl sulfoxide, or a thiyl radical derived from exogenous or endogenous intracellular glutathione, are required to convert proxyl fluorescamine or tempo-9-ac respectively to their fluorescent products.All of the probes were calibrated so that the increase in probe fluorescence after incubation with 2O2 along with 0.2 μM HRP and each probe at its concentration as listed above in Methods. The baseline of the instrument was adjusted to display a relative intensity of zero in the presence of water alone. It is evident from Figure 2O2, all of the probes except for proxyl fluorescamine and tempo-9-ac show relative fluorescence intensities that are less than 5% of the signals they generate in the presence of 1000 nM H2O2 and HRP. With addition of HRP alone, in the absence of exogenously added H2O2, APF, DCFH, and amplex ultrared show small increases in fluorescence intensity which may reflect spontaneous formation of H2O2 but these signals are still less than 20% of the signals generated in the presence of 1000 nM exogenous H2O2 and HRP. The two fluorogenic free radical scavengers, tempo-9-ac and proxyl fluorescamine, however, clearly undergo significantly smaller increases in fluorescence with increasing H2O2 concentration than the other probes. We hypothesize that these smaller increases reflect the indirect mechanisms by which hydroxyl radicals modify the two spin traps , resulting in suboptimal conversion of the probes to the spin-neutral species.In order to determine hydroxyl radical generation from hydrogen peroxide in the presence of HRP or from ferrous iron solutions and particulate suspensions, calibration curves were first produced. In the presence of HRP, the fluorescence intensity of all of the probes increases with increasing hydrogen peroxide concentration Fig. . The gai2O2 and HRP, but the signal from each probe in the absence of H2O2 and HRP , rather than the signal from water alone, was set to a relative fluorescence intensity of zero.In order to measure hydroxyl radicals generated by ferrous iron solutions and pyrite suspensions, a different calibration curve was used. The gain was still adjusted to display a relative fluorescence intensity of 1000 in the presence of 1000 nM H2O2 to the fluorescent resorufin in the presence of HRP. It is evident that H2O2 is indeed formed in addition to hydroxyl radicals upon addition of ferrous iron salts or minerals because addition of HRP to the incubation mixtures containing the iron samples and amplex ultrared results in a significant increase in fluorescence.In an aqueous solution containing dissolved oxygen, the addition of soluble ferrous salts or ferrous iron-containing minerals such as pyrite results in the formation of both hydrogen peroxide and hydroxyl radicals (eqs 1 and 2). All of the probes were incubated with multiple concentrations of ferrous iron in 25 mM phosphate buffer, pH 7.4  Fe(II), and (3) pyrite suspensions.A major goal of these studies was to evaluate several fluorogenic probes for their sensitivity to hydroxyl radicals, especially at the lower limit of detection, as well as their suitability for use with particulate mineral suspensions. Specifically, we have used the probes to determine the apparent concentrations of hydroxyl radicals generated in a 24 hour time period, at room temperature, in phosphate buffer at pH 7.4. Changing any of these variables would probably lead to different apparent concentrations of hydroxyl radicals being detected. However, by keeping these variables constant, a comparison of all the probes could be performed for three systems: (1) H2O2 and HRP, each probes fluorescence' is different . Because both tempo-9-ac and proxyl fluorescamine will not directly react with H2O2 in the presence of HRP, their responsiveness' is reduced. In the presence of HRP but absence of H2O2, all of the probes exhibit some level of fluorescence. This fluorescence level is higher than the background fluorescence level for each probe /DMSO (for proxyl fluorescamine) as a fluorescence value of 1000. Compared to the fluorescence of only DCFH, APF, and amplex ultrared in a pH buffered water solution, the addition of H2O2 & HRP results in much greater fluorescence intensity. When the fluorometer is calibrated with this large difference in fluorescence intensities, subsequent measurements have a greater accuracy. Both tempo-9-ac and proxyl fluorescamine do not respond with as great of an increase in fluorescence when incubated with H2O2 and HRP. Therefore, the sensitivity of tempo-9-ac and proxyl fluorescamine are lower than the sensitivities of DCFH, APF, and amplex ultrared. The background fluorescence for proxyl fluorescamine is only slightly less than the fluorescence of proxyl fluorescamine reacted with 1 μM H2O2 and HRP . When proxyl fluorescamine is incubated with either Fe(II)  Fig. , the larIf the probes reacted equally with a ferrous iron solution, then they would all detect the same concentration of hydroxyl radicals and all of the curves would overlap. The results shown in Figure 2/L results in a decrease in the detected hydroxyl radical concentration. This may be due to several factors: (1) a decrease in the stability of amplex ultrared in the presence of greater concentrations of hydroxyl radicals, (2) amplex ultrared adsorption to the larger surface areas of pyrite present in higher loadings, (3) an interaction between iron and the probe or one of the reactants , or (4) alteration of solution pH in the presence of higher loadings of pyrite. Although we evaluated pyrite particles for generation of ROS for 24 hours, the time span of incubation with the probes could have been shorter or longer. The probes appear to increase in fluorescence over time when incubated with particles suggesting that the detection of ROS is cumulative.In the presence of pyrite particles Fig. , each prThroughout the study, the solutions containing APF were the easiest to prepare, contained a minimal amount of additional reactants and the results were the easiest to interpret. Unlike DCFH, APF was used directly from the purchased vial without further treatment. Both proxyl fluorescamine and tempo-9-ac required addition of DMSO and glutathione/phenol, respectively. Proxyl fluorescamein and tempo-9-ac also required preparation from powders and quantification using UV-Vis spectroscopy – APF was purchased as a 5 mM solution.The objective of these studies was to evaluate several fluorescence-based techniques for measuring hydroxyl radicals in solution with the ultimate goal of using one or more of the techniques to compare the concentration of hydroxyl radicals that are generated in particulate suspensions. The results show that some probes are more suitable for these goals. APF, DCFH, and amplex ultrared do not require extra reactants other than water and a pH buffer to quantify hydroxyl radicals, which results in their higher sensitivity and ease of use. Proxyl fluorescamine and tempo-9-ac both require other reactants and their use is limited to high precision measurements and solutions with low concentrations of reactants such as ferrous iron and pyrite, respectively. DCFH and amplex ultrared would also be suitable but DCFH requires an additional de-esterification step for its use in cell-free solutions and both DCFH and amplex ultrared will react with ROS other than hydroxyl radicals. APF is both sensitive and selective for only the most highly reactive ROS, including hydroxyl radicals. The fluorogenic probes that were evaluated here were chosen because they can be purchased and used with little further treatment. This study is not an extensive study of all methods available and many other probes are available for the detection of ROS; for reviews, see -63.2DCFDA, were used without further treatment. In all of the experiments, the reactant solutions, including mineral particles if used, were combined in 2-mL centrifuge tubes at room temperature (22 ± 2°C). The tubes were placed on an end-over-end orbital mixer in the dark to prevent photo-activated oxidation. After 24 hours, particulate suspensions were filtered through Millipore PVDF 0.45 micron filters and, subsequently, transferred to 4-mL methylcrylate fluorescence cuvettes prior to fluorescence measurements using the appropriate wavelength filters. The filtration step was omitted in experiments conducted without particles.The methods for measurement of hydroxyl radicals using the different probes were all performed in a similar fashion. All of the probes were purchased from Invitrogen and, except for H2O2 was added to the probe dissolved in 50 mM potassium phosphate buffer at pH 7.40, along with 2.95 units/mL  Sigma type II horseradish peroxidase (HRP). The reaction between H2O2 and horseradish peroxidase oxidizes the probes stoichiometrically, resulting in an increase in probe fluorescence. This method for oxidizing the probe molecules was used instead of employing the Fenton reaction to generate •OH because of the difficulty in controlling the quantity of •OH formed in the Fenton reaction. By using a series of calibration curves generated from known quantities of H2O2 in the presence of HRP, the values of relative fluorescence intensity from probes in the experiments described below could all be converted to corresponding concentrations of •OH (nM). The water used in the experiments  was stored in the dark for at least one month before being used in experiments to limit the background concentration of H2O2.Calibration curves were generated similarly for all of the fluorescent probes. A known amount of Hp-aminophenyl) fluorescein (APF) is not fluorescent until reacted with •OH -3-phenyl-2-pyrrolin-4-one, potassium salt (proxyl fluorescamine) was prepared by dissolving 5 mg of proxyl fluorescamine in 5.125 mL DMSO to obtain a 2 mM proxyl fluorescamine solution. An aliquot of the stock solution was added to reaction vials containing a total volume of 1.7 mL in which components for generation of hydroxyl radicals were present so that the final concentration of proxyl fluorescamine was 100 μM and 5% DMSO was present. Fluorescamine fluorescence was quantitated spectrofluorimetrically with excitation and emission wavelengths of 390 nm and 490 nm, respectively.-1 [4-((9-acridinecarbonyl)amino)-2,2,6,6-tetramethylpiperidin-1-oxyl (tempo-9-ac) has been developed as a fluorogenic spin trap by Borisenko et al.  to serve-1 ). All of2/g was determined using five-point N2 adsorption BET.Natural pyrite  obtained from Ward's Natural Science, Rochester, NY was crushed in an agate mill and was sieved to obtain particle sizes between 38 to 63 μm. The ground pyrite was stored in a vacuum desiccator until used in the experiments. A surface area of roughly 1.25 mCAC designed the study, performed the experiments and drafted the manuscript. SRS participated in the design of the study. MAAS coordinated the study and provided funding. All authors read and approved the final manuscript.


A soft agar colony assay has been developed for the B16 mouse melanoma and the Lewis lung tumour. The special features of the technique are the use of a gas phase with 5% O2 instead of air and the addition of rat red blood cells. Single cell suspensions are prepared by trypsinization from the solid tumour and the cells are plated out in 0-3% agar over a layer of 0-5% agar in 30-mm Petri dishes. After 8 to 15 days' incubation in 5% O2, colonies of more than 50 cells are produced. Plating efficiencies of between 30 and 50% are usually obtained. The addition of up to 10(4) heavily irradiated tumour cells gives some further improvement in plating efficiency for the B16 melanoma but not for the Lewis lung tumour. Applications of the technique to measure cell survival in the two tumours after treatment with cytotoxic drugs and radiation are reported. The scatter of experimental points is relatively small, and in comparative experiments good agreement has been obtained with results using in vivo assay techniques.


Modulation of tumour cell growth by tumour-infiltrating leucocytes is of high importance for the biological behaviour of malignant neoplasms. In melanoma, tumour-associated macrophages (TAM) and tumour-infiltrating lymphocytes (TIL) are of particular interest as inhibitors or enhancers of cell growth. Recruitment of leucocytes from the peripheral blood into the tumour site is mediated predominantly by chemotaxins, particularly by the group of chemokines.−1) was about 5–50 times higher than interleukin 8 (IL-8), determined in the same supernatant samples. Tumour necrosis factor α (TNF-α), not, however, IL-2, interferon-γ (IFN-γ), or α-melanocyte-stimulating hormone (α-MSH) was able to up-regulate RANTES and interleukin 8 secretion. Furthermore, higher levels of RANTES secretion in vitro were associated with increased tumour formation upon S.C. injection of six human melanoma cell lines in nude mice. Our data provide evidence that a subset of melanoma cells express mRNA and secrete RANTES protein which may be partly responsible for the recruitment of monocytes, T-cells and dendritic cells into the tumours. However, transplantation experiments in nude mice suggest that effects of RANTES may also benefit tumour progression. Further studies are needed to dissect the underlying mechanisms. © 1999 Cancer Research CampaignThe aim of this study was to identify peptides released by human melanoma cells with monocyte chemotactic properties. To assure the presence of biologically active mediators, biochemical purification and biological characterization of peptides was based on a detection system dependent on bioactive, monocyte chemotactic activity in vitro. Cell culture supernatants of melanoma cells were fractioned by heparin–sepharose followed by preparative reversed-phase HPLC steps to enrich monocyte chemotactic activity in one single band on a sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel. These purified fractions were shown to react with RANTES-specific antibodies in an enzyme-linked immunosorbent assay (ELISA) as well as in Western blot analysis. Amino acid sequencing of the N-terminal protein fragment confirmed 100% homology to the RANTES protein. Further analysis showed that four out of eight melanoma cell lines constitutively expressed and secreted the β-chemokine RANTES as detected by ELISA. The amount of RANTES protein secreted (up to 50 ng ml


Attempts were made to induce immunity to 5 spontaneous rat sarcomas transplanted into syngeneic recipients. Rats were immunized by surgical removal of growing tumour transplants or by treatment with attenuated tumour, followed by challenge with tumour cells in suspension. Two tumours wee apparently not immunogenic, but a low level of immunity was induced against 2, and weak evidence of immunity was observed with another. Induced immunity was individually specific rather than cross-reactive. It is concluded that, contrary to some reports, some spontaneous animal tumours are immunogenic in the strain of origin.


The reduction in size of the experimental ISIS 130 tumour has been investigated in LOU rats under the influence of increasing doses of cytostatic agents belonging to different classes. External temperatures of tumours as well as rectal temperatures have been measured at the same time, twice daily, during the whole experiment. The greater the decrease in the tumour size after drug administration, the larger was the decrease in external temperature of tumour. The rectal temperatures remained fairly stable, thus differences between the tumour and rectal temperatures increased. A possible correlation between the reduction of tumour size and the decrease of external temperature of tumour has been traced for every cytostatic agent, and the same linear relationship has been found to link these two parameters. The decrease in external temperature of tumour may, moreover, predict the decrease in tumour size within a term of 1-2 days. Measurement of the magnitude of the transient tumour hypothermia of ISIS 130, following chemotherapy, would represent a new method for measuring the efficiency and duration of action of cytostatic agents.


An originally virus-induced, non-producer tumour system has been studied in relation to humoral and cellular cytotoxic responses to transplantation and other immunization techniques. In all experimental groups cytotoxic lymphocytes (CTL) were observed either directly or after mixed culture of lymphocytes and tumour cells (MLTC). Except for C'-dependent cytotoxic antibodies in mice immunized by irradiated cells, no antibody-mediated cytotoxicity was observed. In 2 protocols (transplantation and immunization by mitomycin-treated cells) CTL in vitro were not protective. In a third protocol (immunization by irradiated cells) CTL afforded partial protection and other factors appeared to be involved. The best in vivo protection was induced by immunization consequent on early surgical removal of a small number of transplanted tumour cells. This study provides lines of evidence for the effectiveness of protection supplied by CTL in well-defined conditions. Comparison with other modes of immunization indicated that these conditions were related to the quantity and to the characteristics of antigen involved.


The survival and clonogenic growth (measured in terms of colony forming efficiency (CFE) of intravenously injected (i.v.) Walker (W256) tumour cells in the lungs of rats was greatly enhanced by states of topical and systemic stress induced by the intraperitoneal (i.p.) injection of rats with a single dose of 10(-5)-10(-3) mmol g-1 body weight of adrenaline and other beta-adrenergic agonists, inflammatory agents , convulsive seizures, "tumbling" or physical restraint. Lowering of innate resistance of the host to growth of seeded tumour cells induced by states of topical and systemic stress, and by the addition of an excess of lethally irradiated (LI) tumour cells to i.v. injected intact tumour cells, were all potentiated by treatment of rats with aminophylline, an inhibitor of cyclic AMP phosphodiesterase. Enhancement of tumour growth by systemic stress was inhibited by bilateral total or medullary adrenalectomy and is attributed to the release and actions of endogenous adreno-medullary hormones. Alpha-adrenergic and most non-adrenergic agents administered in maximum tolerated doses did not significantly affect host resistance to tumour growth in the lungs. These findings, correlated with measurements of cyclic AMP in the lungs of normal and stressed rats, suggest that changes in the resistance of the host to tumour growth involve changes in cyclic nucleotide metabolism in the target tissues (tumour bed); possible mechanisms of action of cyclic nucleotides in this respect are discussed.


Overall health and vitality was better in the dietary restriction-fed mice than in the ad libitum-fed mice. Tumour microvessel density (Factor VIII immunostaining) was two-fold less in the dietary restriction mice than in the ad libitum mice, whereas the tumour apoptotic index (TUNEL assay) was three-fold greater in the dietary restriction mice than in the ad libitum mice. CT-2A tumour cell-induced vascularity was also less in the dietary restriction mice than in the ad libitum mice in the in vivo Matrigel plug assay. These findings indicate that dietary restriction inhibited CT-2A growth by reducing angiogenesis and by enhancing apoptosis. Dietary restriction may shift the tumour microenvironment from a proangiogenic to an antiangiogenic state through multiple effects on the tumour cells and the tumour-associated host cells. Our data suggest that moderate dietary restriction may be an effective antiangiogenic therapy for recurrent malignant brain cancers.Diet and lifestyle produce major effects on tumour incidence, prevalence, and natural history. Moderate dietary restriction has long been recognised as a natural therapy that improves health, promotes longevity, and reduces both the incidence and growth of many tumour types. Dietary restriction differs from fasting or starvation by reducing total food and caloric intake without causing nutritional deficiencies. No prior studies have evaluated the responsiveness of malignant brain cancer to dietary restriction. We found that a moderate dietary restriction of 30–40% significantly inhibited the intracerebral growth of the CT-2A syngeneic malignant mouse astrocytoma by almost 80%. The total dietary intake for the British Journal of Cancer (2002) 86, 1615–1621. DOI: 10.1038/sj/bjc/6600298www.bjcancer.comCancer Research UK© 2002  About 35 000 people in the United States are diagnosed each year with primary or secondary brain tumours . The proSeveral studies suggest that differences in diet and lifestyle can have major effects on tumour incidence, prevalence, and natural history . DietaryRous first suggested that DR might inhibit tumour growth by delaying host-mediated tumour vascularisation . Pili etSince neural tissues utilise glucose as the main energy substrate , brain tad libitum (AL) or under restricted conditions (as below). Water was provided AL to all mice. The animal room was maintained at 22±1°C and cotton nesting pads were provided for additional warmth. All animal experiments were carried out with ethical committee approval in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Care Committee. Also, these procedures meet the standards required by the UKCCCR guidelines (Mice of the C57BL/6J strain and the BALBc/J-SCID (severe combined immuno deficiency) strain were obtained from the Jackson laboratory . The mice were propagated in the animal care facility of the department of Biology of Boston College, using animal husbandry conditions described previously  from a C57BL/6J donor mouse were implanted into the right cerebral hemisphere of anaesthetised recipient mice as we recently described . Each mouse was housed singly in a plastic shoe box cage with a filter top and was given a cotton nesting pad for warmth. Dietary restriction was initiated 7 days prior to tumour implantation and was continued for either 11 or 14 days after implantation. Total DR maintains a constant ratio of nutrients to energy, i.e., the average daily food intake (grams) for the AL fed mice was determined every other day and the DR-fed mice were given 70% of that quantity on a daily basis  and embedded in paraffin. Tumours were sectioned at 5 um, stained with haematoxylin and eosin, and examined by light microscopy.2O2-methanol for 30 min and then blocked with 10% normal goat serum in PBA buffer . The sections were treated with rabbit polyclonal antibody directed against human factor VIII-related antigen  followed by a biotinylated anti-rabbit IgG at 1 : 100 dilution . The sections were then treated with avidin-biotin complex followed by 3-3′ diaminobenzidine as substrate for staining according to the manufacturer's directions . The sections were then rinsed three times with PBS (0.01 M phosphate buffer with 0.9% NaCl). Sections were counter stained with methyl green and mounted. Corresponding tissue sections without primary antibody served as negative controls. Microvessel density was quantified by examining areas of vascular hotspots as previously described by After deparaffinisation, rehydration, and washing, the tumour sections were incubated with trypsin at 37°C for 30 min as we recently described for prostate tumours (−1) for 15 min at room temperature and were then washed in PBS. The sections were treated with 3% H2O2 in PBS for 5 min to quench endogenous peroxide activities. The 3′ hydroxy DNA strand breaks were enzymatically labelled with digoxygeninnucleotide via TdT and incubated for 1 h at 37°C. The reaction was terminated with stop buffer according to the manufacturer's protocol. Sections were then treated with anti-degoxygenin peroxidase for 30 min at room temperature, washed, stained with 3-3′ diaminobenzidine substrate, counter stained with hematoxylin, and finally were mounted. Tissue sections of post weaning normal female mouse mammary glands, provided by Oncor, were used as a positive control and staining of a corresponding tissue section without added TdT served as the negative control.Apoptotic cells were detected using the ApopTag in situ detection kit TUNEL   as we previously described (The apoptotic index was expressed as AI%=A×100/(A+C), where A=TUNEL positive cells and C=counter stained unlabelled cells. The tumour sections were scanned at lower magnification (40× and 100×) for nonnecrotic areas and approximately 2000 total cells were counted for each section at higher magnification (400×). The values of the three sections were averaged and the results of three independent CT-2A tumours were analysed.M citrate buffer (pH 6.0). The sections were heated in a microwave oven for 15 min (defrost cycle) and then cooled to room temperature to unmask the PCNA. Sections were then stained by the same procedures as described above except we used 10% horse serum as blocking agent and PCNA mouse monoclonal antibody (Dako) as the primary antibody. Light microscopy (400×) was used to count both PCNA positive proliferating cells and total tumour cells in three non necrotic areas of each tissue section as previously shown (Proliferation index measured the fraction of cells with proliferating cell nuclear antigen (PCNA) staining as we previously described  1 : 2 (v v−1) at 4°C as we recently described . The mice were maintained for another 7 days under the dietary regime at which time they were euthanised and the Matrigel plug with the surrounding skin was removed as we previously described  body weights (g) of the AL and DR mice after 17 days of treatment were 23.6±0.6 and 20.3±0.4, respectively . Total energy intake and body weights dropped after 17 days in the AL-fed group due to increased tumour burden.The total energy intake of the AL-fed group was about 18 Kcal dayc period . The totDry weights of the intracerebral CT-2A tumours were approximately 79.5% lower in the DR-fed mice than in the AL-fed mice  in the CT-2A tumour, we compared the number of TUNEL positive cells (apoptotic index) in the AL-fed and DR-fed mice. The apoptotic index was almost three-fold greater in the DR mice than in the AL mice . No signin vivo Matrigel angiogenesis model represents early events of angiogenesis and tumour progression and is dependent on activation and infiltration of host stromal cells which include monocytes, macrophages, and endothelial cell precursors (in vivo Matrigel model of angiogenesis (Figure 4The We found that a moderate DR of 30–40% significantly reduced angiogenesis and growth of the CT-2A experimental mouse astrocytoma. Moreover, DR enhanced CT-2A cell apoptosis without effecting cell proliferation. Previous studies showed that moderate DR could reduce the growth of histologically diverse non-neural tumours (Despite a 12% reduction in body weight, the DR-fed mice were more active and healthy than the AL fed mice. Keenan and co-workers recently suggested that the AL feeding of sedentary rodents is a form of over feeding that can produce adverse health effects (We found that angiogenic biomarkers may be useful for evaluating the influence of energy intake and nutrition on the growth and progression of experimental brain cancer. Moderate DR significantly reduced microvessel density, increased the apoptotic index, but had little effect on the PCNA labelling index in the CT-2A brain tumour. Other investigators have also reported that antiangiogenic growth factors and cytokines can reduce tumour microvessel density, increase apoptosis, but have little effect on cell proliferation .The mechanisms by which DR reduced CT-2A tumour angiogenesis and growth are not yet clear, but may involve effects on both the tumour cells and the tumour-associated host cells. It is documented that human and experimental gliomas are dependent on glycolysis for energy (Glucose is used exclusively for adult brain energy metabolism under normal physiological conditions, but the brain will metabolise ketone bodies for energy when blood glucose levels decrease as during fasting or DR . We receIn addition to possible effects on energy metabolism, DR may also reduce CT-2A angiogenesis and growth through effects on tumour associated host cells. The progression of human and experimental brain tumours is dependent to a large extent on the proangiogenic and inflammatory properties of activated glia and macrophages . Indeed,in vivo Matrigel model of angiogenesis indicating that DR can reduce angiogenesis both within and outside of the central nervous system. It is possible that DR reduces the inflammatory properties of tumour-associated host cells and thereby shifts tumour-host cell interactions from a proangiogenic to an antiangiogenic state. Studies are planned to test these possibilities.We also found that DR caused a noticeable reduction in the number and the dilation of blood vessels in the in vivo studies where food intake and body weight are reduced in conjunction with anticancer therapies or with cancer cachexia. Reduction of energy intake as a covariable of anorexic anticancer therapies may confound interpretation of results (Our findings may have relevance to those  results . AlthougAlthough DR is recognised as a preventative measure for carcinogenisis, it is clear from our findings on the the CT-2A brain tumour that DR is not a preventative intervention since all of the tumours implanted grew despite the 7 day DR pretreatment period. The DR-induced inhibition of CT-2A angiogenesis and growth suggests that DR retards tumour progression. Whether DR would also increase the survival time of CT-2A-tumour bearing mice is not clear. Survival studies are difficult with this rapidly growing brain tumour model since the tumour will grow through the implantation burr hole and then subcutaneously over the skull as we previously described for the EPEN model (In summary, we have demonstrated that DR alone can reduce angiogenesis and growth in an experimental mouse brain tumour. Moreover, the antitumour action of DR likely operates through multiple effects on the tumour cells and on the tumour associated host cells. We contend that our experimental protocol may have therapeutic potential for recurrent human gliomas since the time of surgical tumour resection in humans would be comparable to the time of tumour transplantation in mice. In other words, implementation of DR in the clinic could be most effective immediately following tumour removal and may delay tumour recurrence. Because DR is easy to administer and is devoid of adverse side effects, our preclinical studies suggest that DR or caloric restriction may have efficacy as a non-invasive therapy for recurrent malignant brain cancers.


The immunoprophylactic effects of the methanol extraction residue (MER) of BCG were investigated in Strain 2 guinea-pigs injected with cells of the transplantable, diethylnitrosamine-induced, Line 10 hepatocarcinoma. Pretreatment with MER at times ranging from 18 to 182 days prior to tumour implantation protected approximately 40% of guinea-pigs from progressive neoplastic disease. In addition, MER-treated animals developed specific cell-mediated anti-tumour immunity both more rapidly and at higher levels than did non-MER-treated tumour-bearing controls. It was not possible, however, to prognosticate from the results of such laboratory studies to the outcome of immunoprophylaxis.


Using a direct radioimmune antiglobulin technique and a competitive double-antibody radioimmune assay, we have demonstrated the presence of appreciable amounts of host immunoglobulins on the surface and in extracts of cell suspensions from freshly excised solid tumours. IgA appeared to have the greatest concentrations from freshly excised solid tumours. IgA appeared to have the greatest concentration, followed in turn by IgM congruent to IgG2a greater than IgG1 congruent to IgG2b greater than IgG3. The amount of immunoglobulin appeared to be influenced by the tumour under investigation and its mode of maintenance. It could also be increased by the administration of C. parvum but was not significantly influenced by the T-cell status of the host.


We analysed 973 patients with stage I testicular tumours presenting between 1983 and 1994. The median ages at presentation for non-seminomatous germ cell tumour (teratoma) were 27 years, seminoma 36 years and combined tumour 33 years. These differences were statistically significant (Mann-Whitney P < 0.05), suggesting that combined tumours may have a separate natural history. We, therefore, analysed all stage I patients managed with surveillance  post orchidectomy. The actuarial 5 year relapse-free survival and anatomical patterns of relapse were identical for non-seminomatous germ cell tumour (NSGCT) and combined tumour and both were statistically distinct from seminoma . The association of seminoma within a histologically confirmed NSGCT has no influence on the clinical outcome.


Subcutaneous growth of immunogenic chemically induced rat sarcomata and a hepatoma was restricted when cells were injected into syngeneic animals in admixture with MER. Rats rejecting mixed inocula were immune to further challenge with the same tumour. Growth of a chemically induced mammary carcinoma which lacks detectable immunogenicity was suppressed when low cell inocula were injected in admixture with MER or intact BCG organisms, although animals were not immune to re-challenge. These studies indicate that clinically MER may be a suitable alternative to BCG for contact suppression of tumour growth or incorporation into tumour cell:adjuvant vaccines for active immunotherapy.


C-type viruses were formed in heterotransplants of 5/14 human urogenital tumours which had been serially transferred in nude mice of NIH(S) background. Except for one case in which C-type particles were present in the epithelial cells as well as the connective tissue, the viruses were only found within the stroma of the heterotransplanted tumours. Peroxidase labelling with anti-mouse serum demonstrated that the connective tissue supporting the transplanted human tumours was of mouse origin. Competition radioimmunoassays demonstrated that MuLV interspecies viral protein was present in high titre in the transplanted tumour extracts and also in extracts of 2 spontaneous mouse-tumour extracts. These data suggest that endogenous viruses of the nude mice are activated by the graft, and only subsequently infect the human tumour cells and form particles.


Metastatic colorectal cancer is usually progressive despite infiltration of the tumours by T lymphocytes, suggesting that these tumour-infiltrating lymphocytes (TILs) are functionally deficient. Recently, TILs from other tumours have been shown to express reduced levels of the T-cell receptor signal-transducing CD3-zeta chain. We were interested to determine whether a similar abnormality existed in TILs from human colorectal hepatic metastasis (CHM) and, if so, whether correcting the abnormality in vitro would restore anti-tumour activity and provide support for the development of immunotherapy for colorectal hepatic metastases. Twelve of 19 TILs from colorectal hepatic metastases were successfully expanded in vitro in high-dose recombinant interleukin 2 (rlL-2) and their specific anti-tumour cytolytic activity was determined. CD3-positive (CD3+) TILs were HLA-Drhigh and CD69high, suggesting that they had been activated by exposure to antigen but expressed low levels of CD25, CD71 and the nuclear proliferation antigen Ki-67. Furthermore, they showed reduced expression of CD3-zeta compared with autologous peripheral blood T cells (PBTs) and failed to proliferate in the absence of high-dose rIL-2. Expansion of TILs in rIL-2 resulted in restoration of CD3-zeta expression and the ability to lyse K562 and Daudi cells but not autologous tumour cells. The absence of autologous tumour-specific cytolytic T-cell (CTL) activity may be due to the poor immunogenicity of colorectal tumour cells, which we found expressed only low levels of MHC I antigens and CD54 and failed to express MHC II antigens or the co-stimulatory molecules CD80, CD86 or CD106. The inability of rIL-2 to generate tumour-specific CTLs despite restoration of CD3-zeta expression and the presence of an intact lytic mechanism suggests that successful immunotherapy may require the development of strategies to increase the immunogenicity of this tumour.


The tumour blood flow inhibitor 5,6-dimethylxanthenone-4-acetic acid (DMXAA) causes dramatic haemorrhagic necrosis in murine tumours, but activity is seen only at doses close to the toxic limit. This study investigates two approaches for increasing the therapeutic ratio of DMXAA. The first approach combines DMXAA with a second tumour blood flow inhibitor, 5-hydroxytryptamine (5-HT). Co-administration of 5-HT (700 micromol kg(-1)) to C3H mice caused marked enhancement of DMXAA effects against MDAH-MCa-4 tumours, with dose-modifying factors (DMFs) of >3 for blood flow inhibition (at 4 h), 2.3 for necrosis (at 12 h) and 2.0 for growth delay, without compromising the maximum tolerated dose of DMXAA (90 micromol kg(-1)). The data are consistent with ischaemic injury to the tumour being the major mechanism of anti-tumour activity. The second approach combines DMXAA (+/- 5-HT) with hypoxia-selective bioreductive drugs. Anti-tumour activity of all three bioreductive drugs tested  was strongly potentiated by DMXAA, suggesting that there is a population of reversibly hypoxic tumour cells after DMXAA treatment. Co-administration of 5-HT further potentiated anti-tumour activity, but also increased host toxicity of tirapazamine and CI-1010 so that little therapeutic benefit was achieved. In contrast, the host toxicity of the dinitrobenzamide mustard SN 23816 was only slightly increased by DMXAA/5-HT, whereas the tumour growth delay at the maximum tolerated dose of SN 23816 was increased from 3.5 to 26.5 days. This study demonstrates that 5-HT and/or bioreductive drugs can improve the therapeutic activity of DMXAA in mice, and that with SN 23816 both approaches can be used together to provide considerably enhanced anti-tumour activity.


After tumour excision, anti-TSTA antibody was readily measurable in both serum and lymph. In contrast, the tumour-bearing animal had no measurable anti-TSTA antibody in the serum but low titres in the lymph. Consequently, we formed the hypothesis that in the presence of a growing tumour the serum contained antigen-antibody complexes with antigen in excess.Antibodies to the tumour-specific transplantation type antigen (TSTA) of a transplanted methylcholanthrene-induced sarcoma (MC-1) in syngeneic rats were studied using the techniques of indirect membrane immunofluorescence and mixed haemadsorption with a To test this hypothesis, tumour-bearing serum was examined for the presence of free antigen and antigen-antibody complexes by 2 different methods. In the first method, tumour-bearing serum was cross-linked with glutaraldehyde and was found to absorb specifically the anti-TSTA antibody, indicating free circulating TSTA. Next, antigen-antibody complexes were split with salt or acid and separated into a low molecular weight (or “antigen”) fraction  and a high molecular weight (or “antibody”) fraction . The low M.W. fraction specifically inhibited the anti-TSTA antibody when tested by either membrane immunofluorescence or mixed haemadsorption, indicating the presence of antigen from antigen-antibody complexes in the tumour-bearing circulation. The possible effect on the host's immune response of circulating free tumour antigen and antigen-antibody complexes are discussed.


Intravenous injection of Corynebacterium parvum (C. parvum) 4 days after s.c. inoculation of 5 X 10(5) cells derived from the immunogenic fibrosarcoma FSA/R induced tumour growth inhibition over a period of 21 days in syngeneic C3H/Buf mice. This was not accompanied by a change in the proportions of host cells within the tumour, but the activation state of tumour-infiltrating macrophages was increased following C. parvum therapy. Two macrophage subpopulations were identified in FSA/R tumours after fractionation by unit gravity velocity sedimentation. After i.v. C. parvum therapy the tumour-infiltrating macrophage subpopulation which sedimented between 1 and 6 mm h-1 was consistently activated as determined by measurement of Fc receptor avidity. Other intra-tumour macrophages were generally unaffected by C. parvum treatment. We have previously shown that the host cell fraction sedimenting between 1 and 6 mm h-1 is enriched with monocytes and the data presented in this paper suggest that these cells may enter the tumour in a pre-activated state following intravenous C. parvum therapy.


This paper explores the factors involved in assessing the value of a tumour-marker test in differential diagnosis and patient monitoring. The difficulties have been grossly underestimated, and in the past it has been tacitly assumed that an association between the tumour-marker level and the presence of malignancy is sufficient to prove the usefulness of the test. Part 1 investigates in depth the principles of design of a study to evaluate a tumour-marker test, bearing in mind the ultimate aim of improving the patient's prognosis. The preliminary research carried out on the tumour marker, the laboratory assay technique itself and the "normal" range of levels or the use of a proposed critical level, are all reviewed. The clinical side presents many problems, including the precise definition of the medical situation in which the addition of a new test may assist, and the estimation of the overall size of this problem. Some examples from studies are given. The general principle of evaluating a tumour marker by considering the clinical situation first without, and then with, a tumour-marker result, is stressed. Part 2 gives some practical advice on the setting-up, administration and analysis of such studies.


To determine whether tumour growth is influenced by circadian variations in tumour tissue blood flow, we measured changes in area doubling time of tumours (Sato lung carcinoma) within transparent chambers and changes in tissue blood flow of rat subcutaneous tumour during a light-dark cycle. Rats were subjected to an artificial light-dark cycle with light from 7 a.m. to 7 p.m. Tumour doubling times (TDTs) during the dark and the light spans were 33.5 +/- 11.9 h  and 70.6 +/- 36.9 h  respectively. The former was significantly shorter than the latter (P < 0.001). In addition, the larger the tumour became, the longer was the TDT during the light span (P < 0.05). Tumour tissue blood flow during the night (10 p.m.-4 a.m.) was approximately 1.5 times greater than that during the day (10 a.m.-4 p.m.). The time during which tumours actively grow and that during which tissue blood flow in tumours increases coincided. These results strongly suggest that tumour tissue blood flow is a determining influence on tumour proliferative activity and that tumour growth is influenced by circadian variations in tumour tissue blood flow.


Tumour cells from a polyoma-induced ascitic tumour were fractionated on the basis of the electrical charge on the cell surface by free-flow electrophoresis. Several characteristics of tumour cells have been investigated: (1) differences in the proliferation and antigenicity within the tumour at any point in time; (2) variation in proliferative potential with the ageing of the tumour. In early ascitic tumours, electrophoretically fractionated cells exhibit very similar proliferative characteristics. However, most DNA synthesis was found in slow-moving cells. The behaviour of older tumours was different. Proliferative potential and DNA synthesis were weaker and restricted to slow-moving cells, suggesting that fast-moving cells in older tumours were resting cells. An enrichment in immunoglobulin-bearing cells was also found in slow-moving cell fractions, supporting the hypothesis of variability in expression of tumour-specific antigens. The role of cell-surface properties and cell kinetics is discussed in relation to electrical surface charge, which might be involved in cell dissemination and metastasis. Thus, free-flow electrophoresis represents a satisfactory approach to isolate tumour cell subpopulations with characteristics such as high proliferative potential or increased expression of tumour antigens.


The efficacy of three murine monoclonal antibodies (MAbs) for immunoscintigraphy of small-cell lung cancer (SCLS) xenografts was studied in a Balb/c nu/nu mouse model. These Mabs, 123C3, 123A8 and MOC191, belong to cluster 1 of anti-SCLC MAbs and bind to the neural cell adhesion molecule (NCAM) with similar affinity. After intraperitoneal injection of these MAbs, labelled with 125I, the highest uptake in tumour tissue was obtained with MAb 123C3. Seven days after the administration of this MAb 13.9% of the injected dose per gram of tumour tissue was retained in the tumour. The corresponding tumour tissue ratios ranged from 3.97 for blood to 31.03 for colon. The imaging results and the tumour uptake were less favourable for the two other MAbs, 123A8 and MOC191 (fractions of injected dose respectively 6.7% and 9.2%), although affinity, biological activity after labelling and uptake in non-tumour tissues were very similar for all three MAbs. These results may be explained by the differences in the interaction between the MAbs and the tumour cells. Mab 123C3 is internalised into tumour cells, whereas both other anti-NCAM Mabs are not. Internalisation into NCI H69 cells was demonstrated in vitro by radioimmunoassay, confocal laser scanning microscopy and electron microscopy. The internalised fraction of MAb 123C3 was 22.3% after 24h, whereas this fraction was only 7.5% for MAb 123A8. Although the internalised radiolabeled Mabs are usually degraded and dehalogenated intracellularly, the retained radioactivity is high. Apparently, intracellular degradation of radiolabelled MAb 123C3 and subsequent secretion of radioactive iodine did not prevent the accumulation of intracellular radioactivity. In conclusion, accumulation and retention of radioactivity in the tumour tissue, due to internalisation of radiolabelled MAbs, may improve the results immunoscintigraphy.


Drug targeting systems are nanometre-sized carrier materials designed for improving the biodistribution of systemically applied (chemo)therapeutics. Various different tumour-targeted nanomedicines have been evaluated over the years, and clear evidence is currently available for substantial improvement of the therapeutic index of anticancer agents. Here, we briefly summarise the most important targeting systems and strategies, and discuss recent advances and future directions in the development of tumour-targeted nanomedicines. In this review, we briefly address the most important nanomedicine systems and strategies, summarise the clinical status and highlight future directions.Over the past few decades, our knowledge on the aetiology of cancer has increased exponentially . This imTumour-targeted nanomedicines currently in clinical use are shown in Liposomes are frontrunners among the nanomedicine systems developed so far . Liposomμg hml−1 were found in patients, as compared with 2.5 h and 45 μg h ml−1 for Myocet, and with 55 h and 900μg  h ml−1 for Caelyx, respectively  and progression-free survival times (PFS: 4 months for both), the incidence of cardiac events (29 vs 13%) and of congestive heart failure (8 vs 2%) were significantly lower for Myocet  of 4 ectively . Howeverr Myocet . In certBesides Myocet and Caelyx, several other liposomal nanomedicines have been evaluated over the years, including, for example, non-PEGylated liposomal daunorubicin (DaunoXome) and vincristine (Onco-TCS), PEGylated liposomal cisplatin (SPI-77) and lurtotecan (OSI-211), and lipoplexes, such as Allovectin and LErafAON, in which cationic lipids are used to complex, carry, protect and deliver genetic material, such as plasmid DNA and antisense oligonucleotides . ThermodTen years after the first report on liposomes , naturalN-(2-(hydroxypropyl)methacrylamide)) through an enzymatically cleavable tetrapeptide spacer (GFLG). Like Myocet and Caelyx, PK1 primarily improved the therapeutic index of doxorubicin by attenuating its (cardio)toxicity  or the free drug . As compared with Taxol, Abraxane significantly improved both the response rate (33 vs 19%) and the PFS time (23 vs 17 weeks) of systemic taxane treatment (vs 22%), despite the 50% higher dose, and no hypersensitivity reactions were observed, despite the absence of premedication  was the first passively tumour-targeted polymeric nanomedicine to gain Food and Drug Administration (FDA) approval. Evidence for an advantage of Abraxane over the standard, Cremophor-formulated version of paclitaxel has been provided by a large phase III trial in which >400 women with metastatic breast cancer were randomized to receive either Abraxane , has been used for treating patients with acute lymphoblastic leukemia for >10 years; Zinostatin, that is, PSMA-bound neocarcinostatin, has been approved in Japan for the treatment of liver cancer; and Xyotax, that is, PLGA-conjugated paclitaxel, is in phase III evaluation for ovarian and non-small-cell lung cancer. In addition to such conventional polymer–drug, polymer–protein and protein–drug conjugates, several novel types of polymeric nanomedicines have also recently entered clinical trials, including cationic polyplexes for DNA and siRNA delivery  expressed by cancer cells, for instance, the observed improvements in antitumour efficacy were found to be due to an enhanced cellular internalisation of the agents, rather than to an increased tumour accumulation . In addiβ3, αvβ3 and α5β1, and through aminopeptidase-N, respectively. Recent data obtained in our group with RGD-targeted liposomes containing vascular targeting agents showed widespread central necrosis in established experimental tumours . Opposite to the short-lasting antitumour effects obtained with the free agent, endothelial cell-targeted liposomal delivery halted tumour progression for significantly prolonged periods of time. Further preclinical and clinical studies on the efficacy of tumour vasculature-targeted nanomedicines are eagerly awaited. If such nanomedicines ultimately prove in the clinic to be efficacious with manageable side effects, combination therapies together with radiation, chemotherapeutic agents and/or antiangiogenic drugs are anticipated to attack the thin film of viable tumour cells in the periphery of the tumour, which usually survives when vascular targeting agents are applied as anticancer therapeutics.Destruction of the endothelium in solid tumours can result in the death of tumour cells induced by the lack of oxygen and nutrients. This observation, together with the high accessibility of luminal surface receptors, has led to the design of nanomedicines actively targeted to tumour endothelial cells . LigandsIn this review, we have primarily restricted ourselves to tumour-targeted nanomedicines designed for the improved delivery of already established, low-molecular-weight chemotherapeutics. Many of the new drugs arising from advances in biotechnology, however, are macromolecules, such as proteins and nucleic acids. The clinical development of these challenging and often fragile molecules will likely also profit substantially from the attributes of targeted nanomedicines, providing these complex molecules, for example, with protection against degradation and elimination, and with improved access to target cells and tissues.in vivo fate and performance of targeted nanomedicines is currently receiving intense attention, and will certainly facilitate the implementation of imaging-guided drug delivery to promote the optimal use of (tumour-) targeted nanomedicines.In the document ‘Forward Look on Nanomedicine’, the European Science Foundation included in their definition of the discipline of nanomedicine not only the use of nanometer-sized materials for the treatment but also for the diagnosis of diseases. Regarding the latter aspect, the development of high-resolution imaging techniques  for the rapid, noninvasive monitoring of the the design of systems that are able to respond to externally applied stimuli, such as, hyperthermia, ultrasound, light and magnetic fields, and that can be triggered to release their contents  to inhibit tumour-associated inflammation  drug targeting  chemotherapy.Additional areas likely to receive considerable attention in the years to come are:For obvious reasons, the latter of the above strategies has thus far received the most clinical attention. During surgery, for instance, sustained-release delivery devices, such as Gliadel , can be implanted into those parts of glioblastoma lesions that cannot be removed surgically see . In addiSince the approval, in 1995, of the first tumour-targeted anticancer nanomedicine , targeted nanomedicines have become an established addition to the anticancer drug arsenal, with several formulations presently on the market. A major limitation impeding the entry of targeted nanomedicines onto the market is that new concepts and innovative research ideas within academia are not being developed and exploited in collaboration with the pharmaceutical industry. An integrated ‘bench-to-clinic’ approach, realised within a structural collaboration between industry and academia, would strongly stimulate the progression of tumour-targeted nanomedicines towards clinical application.


Vincristine pharmacokinetic, tumour uptake and therapeutic characteristics were investigated here in order to elucidate the processes underlying the enhanced efficacy observed for vincristine entrapped in small (120 nm) distearoylphosphatidylcholine/cholesterol liposomes. Plasma vincristine levels after intravenous (i.v.) injection are elevated more than 100-fold in the liposomal formulation compared with free drug in tumour-bearing as well as non-tumour-bearing mice over 24 h. Biodistribution studies demonstrate that the extent and duration of tumour exposure to vincristine is dramatically improved when the drug is administered i.v. in liposomal form. Specifically, 72 h trapezoidal area under the curve values for liposomal vincristine in the murine L1210 ascitic and B16/BL6 solid tumours are 12.9- to 4.1-fold larger, respectively, than observed for free drug. Similar to previous results with the L1210 model, increased drug delivery to the B16 tumour results in significant inhibition of tumour growth, whereas no anti-tumour activity is observed with free vincristine. Comparisons of drug and liposomal lipid accumulation in tumour and muscle tissue indicate that the enhanced efficacy of liposomal vincristine is related predominantly to drug delivered by liposomes to the tumour site rather than drug released from liposomes in the circulation. Consequently, improvements in liposomal vincristine formulations must focus on factors that increase uptake of liposomes into tumour sites as well as enhance liposomal drug retention in the circulation.


Polycystic kidney disease (PKD) is one of the most common genetic diseases in humans. We contend that it may be an emerging infectious disease and/or microbial toxicosis in a vulnerable human subpopulation. Use of a differential activation protocol for the Limulus amebocyte lysate (LAL) assay showed bacterial endotoxin and fungal (1-->3)-beta-D-glucans in cyst fluids from human kidneys with PKD. Fatty acid analysis of cyst fluid confirmed the presence of 3-hydroxy fatty acids characteristic of endotoxin. Tissue and cyst fluid from three PKD patients were examined for fungal components. Serologic tests showed Fusarium, Aspergillus, and Candida antigens. IgE, but not IgG, reactive with Fusarium and Candida were also detected in cyst fluid. Fungal DNA was detected in kidney tissue and cyst fluid from these three PKD patients, but not in healthy human kidney tissue. We examine the intertwined nature of the actions of endotoxin and fungal components, sphingolipid biology in PKD, the structure of PKD gene products, infections, and integrity of gut function to establish a mechanistic hypothesis for microbial provocation of human cystic disease. Proof of this hypothesis will require identification of the microbes and microbial components involved and multifaceted studies of PKD cell biology.


This study tested the therapeutic efficacy of doxorubicin hydrochloride in two formulations: free in saline suspension and encapsulated in polyethylene glycol-coated, long-circulating liposomes. The drug formulations at a dose level of 3 mg doxorubicin per kg body weight were injected intravenously to treat the human pancreatic carcinoma AsPC-1, implanted s.c. into nude Swiss mice. Liposome-encapsulated doxorubicin was significantly more effective in inhibiting tumour growth and in effecting cures, and had only minor systemic toxic side-effects, indicated by a transient weight loss. Confocal laser scanning microscopy was used to determine the tumour uptake and the clearance of doxorubicin in the free and in the liposomal forms. The liposome-encapsulated doxorubicin entered the tumour in greater quantity, and remained in the tumour longer, than the free drug. The liposome formulation produced a sixfold or greater increase in doxorubicin at the disease site. It is probable that increased penetration into the tumour, and long presence with slow drug release from liposomes in the tumour, account for the enhanced therapeutic effect when the drug was encapsulated in polyethylene glycol-coated liposomes.


Delayed-type hypersensitivity (DTH) to Rauscher murine leukaemia virus (R-MuLV) encoded or induced determinants was induced in mice by three syngeneic R-MuLV-induced tumour cell lines, i.e. a myeloid tumour, RMB-1, an erythroid tumour, RED-1, and a lymphoid tumour, RLD-1. DTH to subcutaneously (s.c.) administered RMB-1 cells appeared on day 4, with a maximum DTH response on day 6 or 7. The induction of DTH could be prevented by intravenous (i.v.) pre-immunisation with R-MuLV-induced tumour cells several days before the s.c. immunisation. The three R-MuLV-induced tumour cell lines showed cross-reactivity in the DTH assay, whereas no cross-reactivity was found with syngeneic WEHI-3 cells. This indicates that the three R-MuLV-induced tumour cell lines share a virally encoded or induced antigenic determinant, which activates T-cells. When the RMB-1 cells used for immunisation had been cultured in medium supplemented with interferon-gamma (IFN-gamma), the subsequent DTH response was increased. This coincided with an increased expression of the R-MuLV-specific antigenic determinants on RMB-1 cells as demonstrated by Scatchard analysis. Furthermore, IFN-gamma increased the MHC class I antigen expression on RMB-1 cells, whereas the class II antigen expression remained undetectable.


In primary rhabdomyosarcomata, induced in the rat by intramuscular implantation of powdered metallic nickel, cobalt and cadmium, at least 50% of the high content of inducing metal that accumulates in the nuclei of each tumour, is bound by the nucleoli. The remainder is distributed approximately equally between the nuclear sap and chromatin.


The therapy was continued for more than 6 years, and no progression of the disease was observed. During this period, some histological and immunohistochemical evaluations of the tumour  were performed. Furthermore, the expression of m-RNA for luteinizing-hormone releasing hormone (LHRH) receptors was determined. The results showed a discrepancy between some biological parameters of the tumour and its clinical characteristics. In fact, despite features suggestive of a progression of the cancer (such as the increase of both tumour grading and proliferating capacity (MIB-1), and a fall in the reparative process  being detected, neither local invasion nor metastatic lesions were clinically observed. This discrepancy might be due to the maintenance of high levels of E-cadhezin. Moreover, since this tumour was shown to express mRNA for LHRH receptors, new evidence is provided about the favourable impact of LHRH analogue treatment in patients affected by endometrial cancer. © 2001 Cancer Research Campaign


Caspase-3 activity of the ZnPP IX-treated hepatoma cells increased significantly. Moreover, ZnPP IX-induced apoptosis was completely inhibited by simultaneous incubation with a specific caspase-3 inhibitor and was partially abrogated by bilirubin, a reaction product of HO. In vivo ZnPP IX treatment did not affect nitric oxide (NO) production and tumour blood flow. Western blot analyses showed that HO-1 expression in AH136B cells was strongly upregulated by NO donors, for example, S-nitroso-N-acetyl penicillamine and propylamine NONOate in vitro; conversely, it was remarkably reduced in vivo by pharmacological blockade of NOS. We conclude that HO-1 may function in antiapoptotic defense of the tumour, and thus it may have important protective and beneficial effects for tumour cells against oxidative stress induced by NO, which is produced in excess during solid tumour growth in vivo.Induction of haem oxygenase-1 (HO-1) may provide an important protective effect for cells against oxidative stress. Here, we investigated the mechanism of cytoprotection of HO-1 in solid tumour with a focus on the antiapoptotic activity of HO-1. Treatment of rat hepatoma AH136B cells with the HO inhibitor zinc protoporphyrin IX (ZnPP IX) or tin protoporphyrin IX resulted in extensive apoptotic changes of tumour cells both  Haem oxygenase (HO) is the rate-limiting enzyme in haem degradation; this reaction produces biliverdin, which is subsequently converted to bilirubin by biliverdin reductase in biological systems . Haem oxWe previously showed in an experimental solid tumour that NO produced in excess in the local area surrounding tumour cells seemed to sustain rapid tumour growth  cells were implanted subcutaneously (s.c.) in a dorsal site on the foot of male Donryu rats weighing 160–180 g  with an inoculum size of 1 × 10in vivo, 500 μg kg−1 ZnPP IX  or copper protoporphyrin IX  (500 μg of each in 1 ml of 10% dimethyl sulphoxide (DMSO) in 0.01 M NaOH) was injected intra-arterially (i.a.) into solid tumour via the common iliac artery that is mainly feeding the tumour, so that effective delivery of these compounds is achieved. Solid tumours were obtained 24 h after injection and were stored at −80°C until use for terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labelling (TUNEL) staining and for analyses of HO-1 and HSP70.For the treatment of AH136B tumour 5 cells per well of a six-well polystyrene plate , were incubated with various concentrations of the HO inhibitors ZnPP IX and tin protoporphyrin IX  (−1) fetal bovine serum and 0.5% nonessential amino acids (Invitrogen). After 24 h of culture, the cells were subjected to the cell viability and apoptosis assays of TUNEL staining and caspase-3 activity determination. Similarly, the cells were treated with CuPP IX, which has no direct inhibitory effect on HO activity in vivo   in Dulbe in vivo  and is aNω-nitro-L-arginine methyl ester  or S-methylisothiourea sulphate  in 0.2 ml of 0.9% NaCl solution, given intraperitoneally (i.p.) at a dose of 6 mg kg−1 per day for 5 days, beginning 9 days after tumour implantation. Solid tumour tissues were obtained 1 day after the last injection of NOS inhibitor and were stored at −80°C until use for HO-1 and HSP70 Western blotting. The effect of ischaemic stress on the expression of HSP70 protein in solid tumour was also examined. Briefly, on day 14 after tumour implantation, solid tumours were resected for Western blot analysis at different times after initiation of surgical occlusion of the common iliac artery, which serves the tumour-feeding artery of the tumour-implanted side. In addition, the effect of NO on the expression of HO-1 and HSP70 was examined with the use of AH136B cells in culture treated with the NO donors S-nitroso-N-acetyl penicillamine  and propylamine NONOate (CH3N[N(O)NO]−(CH2)3NH2+CH3) . Western blotting was used to analyse the levels of HO-1 and HSP70 proteins in lysate of the AH136B cells, after a 6-h incubation with the NO donors.Tumour-bearing rats were administered either μm-thick frozen tissue sections were prepared with a cryostat and were air-dried overnight. Apoptotic cells were detected via the TUNEL method by using an apoptosis detection kit  -NH2 fluorescent substrate (Peptide Institute). Ac-DMQD-CHO, caspase-3 inhibitor (Peptide Institute), was added to the reaction mixture at a concentration of 10 μM. Caspase-like amidolytic activity for the peptidyl substrate was measured fluorometrically and caspase activity was determined by subtracting the fluorescence values obtained in the presence of inhibitor.Caspase-3 activity in AH136B tumour cells was measured as described previously  was quantified spectroscopically  was implanted in the solid tumour, and was perfused using a microsyringe pump . After an equilibration period (1 h), dialysate was collected in polyethylene tubes for 15 min, followed by measurement of NO2− and NO3− by using an NOx analyzer   was used for measurement of the tumour blood flow via a probe needle placed into the tumour tissue as described earlier  of tumour tissues treated with ZnPP IX , NOS inhibitors, or vehicle was subjected to Western blot analysis for HSP70. The microsomal fraction obtained by ultracentrifugation was used for the HO-1 analysis. Total protein (25 μg each) in cell lysates or tissue homogenates was used for the Western blotting with a monoclonal antibody to HSP70 , which specifically recognises the inducible but not the constitutive isoforms of the HSP70 family in mammals, or a polyclonal antibody to HO-1 . The protein band that reacted immunologically with the antibody was visualised by using the ECL system .Lysates of AH136B cells treated or untreated with various reagents were prepared as described previously  compared with the control group (14±2 mm−2) and the CuPP IX-treated group (20±4 mm−2)   and the control)  tumour t±4 mm−2) .A significant, although not complete, inhibition of HO activity in solid tumour tissue was obtained by ZnPP IX treatment . The CuPμM CuPP IX  (μM). The viability of tumour cells was inversely decreased in a concentration-dependent manner after ZnPP IX treatment (inset in μM ZnPP IX), illustrating a considerable number of TUNEL-positive cells in the ZnPP IX-treated group compared with the control staining.Cultured AH136B cells were incubated for 24 h with indicated concentrations of ZnPP IX (with or without a caspase-3 inhibitor) or SnPP IX, or with 100 μM ZnPP) . A similμM ZnPP) . The ZnPinset in . Figure μM ZnPP IX, and this increase was totally nullified by simultaneous incubation with caspase-3 inhibitor at concentrations of 1 and 10 μM .We further examined the effect of bilirubin, which is biologically derived from biliverdin, an important enzymatic reaction product of HO . No appreciable suppression for NO production was observed at any time points  after ZnPP IX administration (data not shown).Carbon monoxide (CO) generated by HO has been demonstrated to be a potential endogenous modulator of vascular tone in the liver  to suppress specifically the expression of HO-1. Specifically, endogenous HO-1 mRNA was targeted in cultured SW480 cells, a cell line of human colon adenocarcinoma, by transfecting a 21-nucleotide duplex siRNA derived from the HO-1 nucleotide sequence according to the method reported recently . These data suggest that NO rather than CO derived from HO may be the dominant modulator of tumour blood flow, at least in this solid tumour model.CO has been suggested to function as a neurotransmitter and also as an endogenous modulator of vascular perfusion in the liver through a mechanism analogous to that of NO (2+) released during HO-catalytic decomposition of the porphyrin ring of haem in determining the consequence of HO expression.Although several reports have shown a cytoprotective effect of HO-1 (in vitro and in vivo showed that NO does not participate in HSP70 upregulation in AH136B cells. Thus, we suggest that HSP70 expression in AH136B tumours may be positively regulated by ischaemia or hypoxia through a mechanism different from HO-1 induction involving NO.In our own study, we assumed that some other cytoprotective system may occur to compensate for such a disadvantage of HO-1 induction in our experimental solid tumour. HSP70, which is one of the most important HSPs ubiquitously distributed in mammalian systems (2− and NO3− measurement in our experimental model, indicating that the apoptotic change in the AH136B solid tumours after ZnPP IX treatment depended mostly on the specific suppression of HO activity.AH136B experimental solid tumour tissues produce a high amount of NO, which seems to sustain rapid tumour growth, as we reported previously (in vivo. Thus, HO-1 may become a potential target for cancer chemotherapeutic agents, particularly in combinations with conventional agents. The present study warrants further investigation to develop new tactics for antitumour treatment with the use of HO inhibitors such as ZnPP IX or its polymer-conjugated derivatives with improved pharmacological properties (In conclusion, our current study indicates that HO-1 may function as an antiapoptotic defense system for the tumour, and it may also have important protective and beneficial effects for tumour cells against oxidative stress occurring during rapid growth of solid tumour


Using an under agarose migration (UAM) assay, we studied lymphokine-activated killer (LAK)-attractant activity in cultured conditioned medium of tumour tissues after chemotherapy as a possible mechanism of enhanced LAK cell accumulation into tumour tissues after chemotherapy. BMT-11 is a fibrosarcoma developed in C57BL/6 mice. The conditioned medium of BMT-11 tumour tissues obtained from mice treated with various anti-cancer drugs had chemotactic activity for LAK cells (LAK-attractant activity). mRNA expression of interleukin (IL)-1 alpha, IL-6, IL-8, interferon (IFN)-gamma, and tumour necrosis factor (TNF)-alpha was observed in untreated tumour tissues, which were not enhanced by cyclophosphamide treatment. mRNA expression of TGF-beta 1 was not detected in untreated tumour tissues by reverse transcription-polymerase chain reaction (RT-PCR), but was detected in tumour tissues treated with cyclophosphamide. Recombinant human TGF-beta 1 showed LAK-attractant activity at a concentration of 0.1 ng ml-1 and 1 ng ml-1, whereas fresh splenocytes were not attracted by TGF-beta 1. Anti-TGF-beta 1 antibody inhibited LAK-attractant activity in the conditioned medium of tumour tissues treated with cyclophosphamide to approximately 35% that of control at 100 micrograms ml-1. These findings indicate that TGF-beta 1 produced in the tumour tissues of mice treated with anti-cancer drugs could be a LAK attractant. By a 4 h 51Cr release assay of natural killer cell-resistant BMT-11 tumour cells, we observed that TGF-beta 1 at a concentration from 0.01 ng ml-1 to 10 ng ml-1 did not inhibit LAK activity in an effector phase. Taken together, we suggest that TGF-beta 1 produced in tumour tissues after chemotherapy participates in gathering transferred LAK cells and contributes to the therapeutic effects of transferred LAK cells.


A stathmokinetic method has been used to determine the cell cycle parameters, particularly the potential tumour doubling time, of a murine fat pad sarcoma. Additional information has been obtained by determining the percentage of labelled mitoses (PLM). A technique which simultaneously demonstrates autoradiographically labelled S phase nuclei and histochemically localized acid phosphatase activity has also been used at light microscope level to compare these parameters: acid phosphatase activity was demonstrated in tumour cells and macrophages. Single cell deletion by apoptosis has been investigated as distinct from necrosis. Condensed, dying apoptotic cells, have been found in proliferative areas of tumour that are not under physiological stress. The analysis of apoptosis indicated a previously unsuspected variation in apoptotic activity with tumour weight. Cell death by apoptosis initially rose as the tumour grew, but after the tumour reached a threshold weight it declined dramatically, and finally remained stable. This may reflect an initial attempt at autoregulation of tumour size which ultimately fails. Apoptosis was estimated to account for an average of 7% of the total cell loss rate in this tumour.


These observations are relevant to current proposals for adjuvant immunotherapy of human malignant disease where conditions of minimal residual disease are not being fulfilled.Growth of syngeneic transplants of a 3-methylcholanthrene induced rat sarcoma was suppressed when tumour cells were injected in admixture with BCG. Rejection of these mixed inocula resulted in the suppression of growth of a simultaneous challenge with cells of the same tumour at a contralateral subcutaneous site and conditions for immunotherapy were evaluated with respect to the maximum tumour cell challenge rejected and the optimum time of treatment. These studies established that viable tumour cells were more effective than radiation attenuated cells for the immunizing stimulus. Also, the maximum tumour challenge totally rejected in this way was of the order of 10


Merkel cell carcinoma and dermatofibrosarcoma protuberans are two very rare neoplasms. The simultaneous occurrence of two different tumour entities at the same anatomical site, collision tumours, is a rare phenomenon.We present a rare case of a 74-year-old woman with a previous history of a recurrent dermatofibrosarcoma protuberans presenting with a metastatic Merkel cell carcinoma. Further investigation revealed a collision tumour of a metastatic lesion of the Merkel cell carcinoma within a tumour relapse of a dermatofibrosarcoma protuberans.Synchronous occurrence of two different tumour entities is extremely rare and has not been described for Merkel cell carcinoma and dermatofibrosarcoma. Merkel cell carcinoma, a tumour of the elderly or immunocompromised patients, leads to early metastasis and can be expected to be the limiting factor for prognoses. Primary neuroendocrine tumour of the skin, also known as Merkel cell carcinoma (MCC), was first described by Toker in 1972. It is a rare but aggressive tumour entity of sun-exposed skin of the elderly in the age range of 60 to 90 years. The origin of this tumour is thought to be a line of differentiation according to Merkel cells. Under homeostatic conditions, the Merkel cells, discovered by Friedrich Merkel in 1875, are normal constituents of the basal layer of the epidermis and hair follicles. A higher density of Merkel cells is found in the glabrous epithelium of the digits, lips and oral cavity and hair bearing skin. They represent slow acting mechano-receptors with close contact to unmyelinated nervous fibres in the superficial dermis. Whether these cells are of a neuroendocrine or epidermal-ectodermal nature is still unclear.MCC, with a female predominance,  has a prDermatofibrosarcoma protuberans (DFSP) is another uncommon skin malignancy. As an intermediate neoplasm it has a local aggressive growth pattern but it rarely metastasizes. In the uncommon event of metastasis they are often found in the lungs and in the lymph nodes . A high Clinically, DFSP often presents as slow growing nodular masses of the skin on the trunk or proximal extremities.Simultaneous occurrence of two different tumour entities at the same anatomical site is a very rare event. Cases of haematopoietic neoplasms in association with breast and skin malignancies have been reported. To our knowledge this is the first reported case of a synchronous occurrence (collision tumour) of MCC and DFSP.A 74-year-old, Caucasian woman from Germany with a long history of local recurrences of DFSP on her right upper limb was readmitted for surgical intervention of a tumour of her right axilla. On clinical examination she presented with two firm tumour masses in the right axilla and on the thoracic wall with a size of 6 × 7 cm and 6 × 5 cm. Both lesions displayed central ulceration. In addition, the patient requested the resection of a small (1.7 × 1.6 cm) nodular skin lesion of her right elbow region.The DFSP of her right shoulder region was first diagnosed in 1991. Following several incomplete tumour resections and local progressive tumour growth she was transferred to our hospital in 2001. Tumour resection was carried out and radiation therapy was suggested but refused by the patient. Despite clear resection margins the tumour recurred locally 10 months later and, at this stage, the tumour had infiltrated the brachial plexus. Since then, additional surgical interventions for local recurrences of the DFSP were required every 5 to 9 months. Fifteen years after the initial diagnosis of the DFSP, at readmission for a local recurrence, the tumour extended from the axilla along the thoracic wall to the lateral aspect of the ipsilateral scapular and an additional small (less than 2 cm) reddish blue subcutaneous nodular lesion was observed at the dorsal aspect of the right elbow.Histologically the two tumour masses in the axilla and thoracic wall confirmed the clinical diagnosis of a local recurrence of the known DFSP. The tumours were highly cellular with partly ovoid, partly spindle shaped nuclei with small fascicular cell formations. Due to these clear detectable fascicular areas, these recurrences were classified as DFSP with fibrosarcomatous transformation Figure .1 pN0, pM0 R0).In contrast, the tumour from the elbow microscopically presented as a highly cellular small cell tumour lacking fascicular formations Figure . Additio2b, pN0, pM0, R2) with preservation of the extremity could not be achieved.The Merkel cell carcinoma was resected in total whereas a complete resection of the DFSP  and the relapse tumour of DFSP   Figure . FurtherFor local tumour control, the option of an interthoracoscapular amputation was discussed but not accepted by the patient. Therefore, palliative radiotherapy and chemotherapy with the tyrosine kinase inhibitor Imatinib was initiated.Both DFSP and MCC are very rare malignancies. Even though the occurrence of multiple malignancies in the same anatomical site in organs such as the thyroid, breast and lymph nodes  have beeEven though the association between these two tumours in our patient is most likely coincidental, immunosuppression during a long course of previous tumour disease of the right upper extremity might be accountable for an increased risk of developing a secondary malignancy such as the MCC.Clinical studies and animal experiments demonstrate the fact that malignant tumours lead to an altered immune status systemically, as well as locally, through changes in the microenvironment. Functional impairment of T-lymphocytes in addition to an accumulation of chronically activated myeloid suppressor cells and regulatory T-cells has been observed systemically. This may lead to a disabled tumour killing CD 8 CTL response and enhance cancer development through activation of other immune cells.Changes in local microenvironment, for example an increase in certain pro-inflammatory cytokines and pro-angiogenic mediators such as interleukin 1 and 6  tumour necrosis factor (TNF α) and vascular endothelial growth factor (VEGF), is one mechanism by which the tumour infiltrating leucocytes promote tumour growth, cell survival and an increase in tumour angiogenesis ,8.Many studies have confirmed an increase in certain matrix metalloproteinases (MMPs) altering tissue homeostasis through various biological pathways such as remodelling of the extracellular matrices, increase in cell-cell and cell-matrix adhesion molecules and thereby altering intracellular signalling pathways and often correlating with a poorer prognosis. But many of the mechanisms involved in tumour immunology and tumorigenesis, differentiation and dedifferentiation of cancer stem cells are still unknown, especially mechanisms involved in the synchronous appearance of malignant tumours .For both malignancies described here, surgical resection of the tumour with clear surgical margins is the main choice of treatment. Both tumours are very rare neoplasms with a similar incidence of four cases or less per million per year. DFSP may affect patients of all ages but is rarely encountered during childhood and it mainly occurs in adults between 20 and 40 years of age. It is a tumour of the dermis and subcutis characterized by a local aggressive growth pattern. Its asymmetric growth and pseudopod-like extensions aggravate tumour resection and account for a high rate of local recurrence of up 60% . A stronRadiotherapy has been successful in gaining local tumour control. DFSP has been found to be non-chemotherapy sensitive. Recently the tyrosine kinase inhibitor Imatinib has demonstrated an antitumorigenic effect with promising results for patients with metastatic disease as well as for local tumour control . MCC on In the last 7 years, 12 patients with a MCC were treated in our clinic. Interestingly, four of these 12 patients had a history of an underlying previous malignancy and one additional patient suffered from chronic renal failure. Both conditions possibly lead to a compromised immune response. Compared to other extra pulmonary small cell cancers MCC appears to have a favourable prognosis but it remains an aggressive tumour with a frequent recurrence, early metastases and high mortality rates. In contrast to melanoma, which also represents a neuroendocrine tumour of the skin, tumour thickness seemingly is of no prognostic value. Regional lymph node status appears to influence local recurrence but not the overall prognosis . MCC hasBoth MCC and DFSP are uncommon tumour entities of elderly or immunocompromised patients. Collision tumours are extremely rare and have never been described for MCC and DFSP. In this patient the distant metastasis of the MCC is expected to be the limiting factor for survival.DFSP: dermatofibrosarcoma protuberans; MCC: Merkel cell carcinoma; MMPs: matrix metalloproteinases; VEGF: vascular endothelial growth factor.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.DT conceptualised the case report, gathered the data and wrote the manuscript. ML drafted and revised the manuscript and was involved in the primary surgery. JH gathered the clinical data and assisted with postoperative care of the patient. AD reviewed the literature and assisted in the follow up treatment of the patient. HH performed the primary surgery and took responsibility for the patient's care. HS conceptualised and supervised the process and he gave final approval for publication. CK assessed the histological specimens and drafted and revised the manuscript. All authors read and approved the final manuscript.


The tumour vasculature is vital for the establishment, growth and metastasis of solid tumours. Its physiological properties limit the effectiveness of conventional anti-cancer strategies. Therapeutic approaches directed at the tumour vasculature are reviewed, suggesting the potential of anti-angiogenesis and the targeting of vascular proliferation antigens as cancer treatments.


Previous studies have characterized the enzymatic properties and inhibition of a trypsin-like neutral protease on the surface of Ehrlich ascites cells by means of kinetic analysis. The present study links these kinetic studies with the recently reported role of a tumour-cell membrane-bound serine protease in tumour-induced target cell lysis. Low-mol.-wt inhibitors of this cell-surface trypsin-like neutral protease exhibited a corresponding ability to prevent tumour-induced haemolysis. High-mol.-wt inhibitors of trypsin in free solution had no inhibitory action either on the tumour-bound enzyme or on the ability of tumour cells to lyse erythrocytes. Fragments of tumour-cell membrane retain both the trypsin-like neutral protease activity and the ability for haemolysis. This study represents a correlation between an easily assayed membrane-bound enzyme on tumour cells and a function of possible biological relevance.


The outcome of cancer chemotherapy is determined by an interplay of multiple factors between the host, the tumour, and the drugs administered. Most studies have emphasised the development or selection of drug resistant tumour cells. However, repeated drug treatment of the host may lead to changes  which can influence the subsequent response of the tumour. In this study, we present a model to investigate the role of the host in the development of drug resistance. A drug is administered repeatedly to animals prior to tumour implantation, and tumour response is then evaluated following treatment with the same drug in pretreated and control animals. To illustrate the method, cyclophosphamide was administered weekly for 4 weeks to C3H mice before implantation of the KHT tumour. Tumour growth delay was then compared after one further treatment of cyclophosphamide in this group of animals to that in control mice which had not received the cyclophosphamide pretreatment. Our results indicate that cyclophosphamide produces only a small effect on the host in this system, but the model is a potentially useful one to investigate the contribution of the host in the acquisition of drug resistance.


Noclinical chemistry value was elevated to a magnitude suggesting onset of organ specifictoxicity. However, agents appeared to modulate subpopulations of white blood cells, i.e.,more lymphocytes than PMNs were present in blood from treated animals as determined bydifferential cell counts. This modulation is correlated with increases in granulomatous foci inthe spleen and mesentery of treated animals after 7 days. The kidney was damaged only byCompound 5 at 50 and 100 mg/kg/day; Compound 5 had the most potent anti-neoplasticactivity. The compounds demonstrated no in vitro toxicity against human HCT-8 ileum cells. LD


To study the effect of localised secretion of chemokines on tumour growth, the genes for human (hu) interleukin 8 (IL-8), hu-MCP-1 (MCAF), hu-MIP-1 alpha (LD78), murine (mu)-MCP-1 (JE), mu-MIP-1 alpha or mu-MIP-2 were introduced, via mammalian expression vectors, into Chinese hamster ovary (CHO) cells, and the ability of transfected cells to form tumours in vivo was evaluated. The production of hu-IL-8, hu-MIP-1 alpha or mu-MIP-1 alpha by transfected clones did not influence the growth rate in vitro, but drastically suppressed tumour growth when injected subcutaneously (s.c.) into nude mice. However, clones transfected with hu-MCP-1, mu-MCP-1 or mu-MIP-2 did not show any significant difference in growth rate in vivo compared with clones transfected with vector alone. Histological examination of the site of injection of CHO clones transfected with hu-IL-8, hu-MIP-1 alpha or mu-MIP-1 alpha showed predominantly neutrophilic infiltration. These results indicate that chemokines have potent anti-tumour activity when released, even at low doses, at the tumour site, which may be mediated by recruitment and targeting of neutrophilic granulocytes to chemokine-releasing cells. Our studies highlight the potential usefulness of localised chemokine secretion in inducing potent host anti-tumour defensive responses.


There is a definitive risk of venous air embolism when the fluid infusion is complete and the drip set is still open in a glass bottle. We have devised a novel way of preventing the chances of air embolism when the fluid in the glass bottle finishes. It really gives a “U” turn to the chances of venous air embolism. Air embolism can complicate peripheral IV fluid therapy or centrAir embolism can cause blockage of small vessels in the pulmonary vasculature with compromise of gas exchange, cessation of ventricular pumping caused by blockage of air and arrhythmia, and paraGlass bottles are widely used for administration of intravenous fluids. Various drugs e.g., mannitol are also available only in glass bottles in our part of the world. For the administration of fluids from glass bottles, a needle has to be inserted, which acts as an airway  and the Many times the fluid in the bottle finishes, but the regulating clamp is still open. The needle airway is in place and the infusion line eventually becomes open to air, which can lead to fatal air embolism.For air embolism to occur through an intravenous drip set, there are two prerequisites. First, is that the patient is connected to an intravenous line that is open to air and second is that the intravenous tubing should be placed into a vein whose pressure is below the atmospheric pressure.Sub atmospheric pressure can develop in a vein when the intravenous tubing is attached to the central line, or the vein is on the upper arm in the lateral position. A right-There is a definitive risk of air embolism when the fluid infusion is complete and the drip set is still open.We have devised a novel way of preventing the chances of air embolism when the fluid in the glass bottle finishes. Weix the infusion tubing by giving it a “U” turn so that the loop is below the level of the vein into which the infusion set is attached . This en


Photodynamic therapy (PDT) is an experimental approach to the treatment of neoplasms in which photosensitisers (PSs) accumulated in malignant tissues are photoactivated with appropriate wavelengths of light. The target specificity of PSs may be improved by linking them with carrier macromolecules such as monoclonal antibodies (MAbs). OC125 is a murine MAb that recognises the antigen CA 125, which is expressed on 80% of non-mucinous ovarian tumours. A chlorin derivative conjugated to OC125 was shown to be selectively phototoxic to ovarian cancer and other CA 125-positive cells in vitro and ex vivo. We now report in vivo studies using an ascitic Balb/c nude mouse ovarian cancer model. Ascites was induced by intraperitoneal injection of cells from the human ovarian cancer cell line NIH:OVCAR3. Six weeks after injection, when the animals had developed ascites, biodistribution studies were carried out by injecting the immunoconjugate (IC) or free PS intraperitoneally and sacrificing the animals at 3, 6, 12, 24, 48, 72 and 168 h later. The PS was quantitated by extraction and fluorescence spectroscopy. For both the IC and free PS, peak tumour concentrations were reached at 24 h; however, the absolute concentrations for the IC were always higher (2- to 3-fold) than the free PS. Tumour to non-tumour ratios at 24 h for the IC were 6.8 for blood, 6.5 for liver, 7.2 for kidney, 5.7 for skin and 3.5 for intestine. Evaluation of viable tumour cells in ascites following in vivo PDT with a single light exposure demonstrated a dose-dependent relationship with fluence and IC concentration. However, there was significant treatment-related toxicity at all fluences. With multiple low-dose treatments, the percentage of viable tumour cells was also significantly reduced and there were no treatment-related deaths. These data suggest that, while photoimmunotherapy remains promising as a new treatment modality for ovarian cancers, careful quantitative dosimetry of both IC and light may need to be combined with multiple treatments (as with radiation therapy and chemotherapy) to control malignant disease yet maintain acceptable toxicity in vivo.


The effect of captopril on tumour growth was examined in a xenograft model of human renal cell carcinoma (RCC). Inoculation of the human RCC cell line SN12K-1 (10(6) cells) under the left kidney capsule of severe combined immunodeficient (SCID) mice resulted in the growth of large tumours, with an increase in weight of the inoculated kidney of 3.69+/-1.63-fold (mean+/-s.d.) when compared with the contralateral normal kidney. In mice treated with captopril (19 mg kg(-1) day(-1) or 94 mg kg(-1) day(-1) administered in the drinking water), there was a significant dose-related reduction in tumour development; the tumour bearing kidneys weighed 1.9+/-0.42 and 1.55+/-0.42 times the normal kidneys, respectively . In vitro, captopril at clinically achievable doses (0.1-10 microM) had no significant effect on the incorporation of [3H]thymidine into SN12K-1 cells. Thus, this highly significant attenuation by captopril of in vivo tumour growth does not appear to be due to a direct effect on the proliferation of the tumour cells. Further studies are required to determine the mechanism of inhibition of tumour growth by captopril, in particular to evaluate the role of angiotensin II in this process.


Exposure to the fibrous mineral erionite is known to induce mesothelioma in man and laboratory animals. Previous studies demonstrated the presence of a trypsin-like protease associated with tumour cells. This protease could be demonstrated by the use of fluorescent probes which located cells possessing this enzyme. We have employed this fluorescent probe technique to follow the early events in the lungs of rats exposed to erionite. The evidence presented shows that the mesothelial cells initially lack this enzyme but the enzyme can be detected within hours of exposure of the rat to erionite. The number of mesothelial cells possessing the enzyme steadily increased after a single exposure to the mineral until the animal finally died with a massive pleural tumour. This is the first study of such fluorescent probes in the early stages of tumour induction.