Experimental studies have been carried out using 5-aminolaevulinic acid (ALA) to induce transient porphyrin photosensitisation for photodynamic therapy (PDT) in a pancreatic cancer model in Syrian golden hamsters. ALA was given either intravenously or orally (in bolus or fractionated doses) with the laser light delivered by means of a bare fibre touching the tissue surface or external irradiation using a light-integrating cylindrical applicator. Animals were killed 1-24 h after ALA administration for pharmacokinetic studies and 3-7 days after light exposure to study PDT-induced necrosis. A separate survival study was also performed after a fractionated oral dose of ALA and external irradiation. Protoporphyrin IX sensitisation in the tumour tissue as measured by quantitative fluorescence microscopy was highest after intravenous administration of 200 mg kg-1 ALA and then in decreasing order after oral fractionated and oral bolus doses (both 400 mg kg-1). Laser light application at 630 nm to give 12-50 J from the bare fibre or 50 J cm-2 using surface illumination with the cylindrical applicator resulted in tumour necrosis up to 8 mm in depth. In larger tumours a rim of viable tumour was observed on the side opposite to illumination. In a randomised study, survival of treated animals was significantly longer than in the untreated control group , although all animals died of recurrent tumour. This technique shows promise in the treatment of small volumes of tumour in the pancreas.


Bovine seminal ribonuclease (BSRNase) is an unusual member of the ribonuclease superfamily, because of its remarkable anti-tumour and immunosuppressive properties. We describe here the construction, expression, purification and characterization of a panel of six immunotoxins based upon this enzyme and show that we can increase its anti-tumour activity by over 2 x 10(4)-fold. This is achieved by improving tumour cell targeting using a single-chain Fv (scFv) directed against the oncofetal antigen placental alkaline phosphatase. As well as the simple scFv-BSRNase fusion protein, we have constructed five other derivatives with additional peptides designed to improve folding and intracellular trafficking and delivery. We find that the molecule most cytotoxic to antigen (PLAP)-positive cells in vitro is one that contains a C-terminal 'KDEL' endoplasmic reticulum retention signal and a peptide sequence derived from diphtheria toxin. All these molecules are produced in Escherichia coli (E. coli) as insoluble inclusion bodies and require extensive in vitro processing to recover antigen binding and ribonuclease activity. Despite incomplete ribonuclease activity and quaternary assembly, these molecules are promising reagents for specific chemotherapy of cancer and are potentially less harmful and immunogenic than current immunotoxins.


The inability to assess the extent of tumour damage immediately following treatment is a major clinical obstacle to improving the management of cancer patients. Normally, the effectiveness of chemotherapy or radiation therapy cannot be determined for at least several weeks after treatment. We studied the increase in mRNA of the growth arrest and DNA damage-inducible gene GADD153 in human 2008 ovarian carcinoma cells in vitro and in vivo to determine whether treatment-induced increases in the level of GADD153 mRNA could be used as a marker of the extent of tumour damage. GADD153 mRNA was increased in a transient, dose-dependent manner by cisplatin (DDP) when the tumour cells were grown both in vitro and as tumour xenografts in nude mice. The magnitude of induction of GADD153 mRNA did not vary significantly between different 2008 xenografts treated with equal doses of DDP, and GADD153 mRNA induction correlated with the degree of in vitro cytotoxicity for two different schedules of drug exposure. DDP increased GADD153 mRNA levels in melanoma and head and neck xenograft models as well. We conclude that the increase in GADD153 mRNA can be used to detect tumour injury at time points as short as 24 h after administration of DDP.


Immunological therapies enhance the ability of the immune system to recognise and destroy cancer cells via selective killing mechanisms. DNA vaccines have potential to activate the immune system against specific antigens, with accompanying potent immunological adjuvant effects from unmethylated CpG motifs as on prokaryotic DNA. We investigated an electroporation driven plasmid DNA vaccination strategy in animal models for treatment of prostate cancer.PSA gene (phPSA) was delivered in vivo by intra-muscular electroporation, to induce effective anti-tumour immune responses against prostate antigen expressing tumours. Groups of male C57 BL/6 mice received intra-muscular injections of phPSA plasmid. For phPSA delivery, quadriceps muscle was injected with 50 μg plasmid. After 80 seconds, square-wave pulses were administered in sequence using a custom designed pulse generator and acustom-designed applicator with 2 needles placed through the skin central to the muscle. To determine an optimum treatment regimen, three different vaccination schedules were investigated. In a separate experiment, the immune potential of the phPSA vaccine was further enhanced with co- administration of synthetic CpG rich oligonucleotides. One week after last vaccination, the mice were challenged subcutaneously with TRAMPC1/hPSA (prostate cancer cell line stably expressing human PSA) and tumour growth was monitored. Serum from animals was examined by ELISA for anti-hPSA antibodies and for IFNγ. Histological assessment of the tumours was also carried out. In vivo and in vitro cytotoxicity assays were performed with splenocytes from treated mice.Plasmid expressing human The phPSA vaccine therapy significantly delayed the appearance of tumours and resulted in prolonged survival of the animals. Four-dose vaccination regimen provided optimal immunological effects. Co - administration of the synthetic CpG with phPSA increased anti-tumour responses, preventing tumour occurrence in 54% of treated animals. Vaccination with phPSA resulted in anti-hPSA Abs production and a significant production of IFNγ was observed in immunised animals (p < 0.05). Immune responses were tumour specific and were transferable in adoptive T cell transfer experiments.In vivo electroporation mediated vaccination is a safe and effective modality for the treatment of prostate cancer and has a potential to be used as a neo-adjuvant or adjuvant therapy.This phPSA plasmid electroporation vaccination strategy can effectively activate tumour specific immune responses. Optimisation of the approach indicated that a four-dose regimen provided highest tumour protection.  Prostate cancer remains a major health issue in the present era, largely due to limitation of therapeutic options especially in advanced disease. Prostate cancer represents the most common non-cutaneous cancer and is the second leading cause of cancer related deaths among American men . There aal. [+ T-cell immune responses have been reported in patients with advanced prostate cancer [Healthy individuals are known to have some immune inhibitory effects on prostate cancer growth (at least early phase of the disease), while progressive tumour development is a result of tumour escape from the immune system. Many factors are involved in tumour immune escape. Blades et al.  have shoal. , abnormaal.  and expral. . Immunolal. . Both ae cancer -10.in vivo DNA plasmid vaccination, in terms of dose schedule and combination with CpG oligonucleotides. We investigated the utilisation of a human PSA expressing plasmid in a murine model of prostate cancer. PSA, a serine protease secreted by both normal and transformed epithelial cells, is almost exclusively expressed on prostatic epithelial cells, and its expression is conserved in nearly all advanced prostate cancer [For malignant diseases different approaches of active immunisation have been explored, including vaccination with cDNA , RNA 1, proteine cancer . PSA is e cancer . Althouge cancer . Additioe cancer .DNA vaccines induce immune responses by direct expression of the antigen by the host cells. Electric pulse parameters optimal for the plasmid delivery have been shown to enhance humoral immune responses . MoreovehPSA) expressing plasmid pUMFG/PSA/IRES/CD25 (hereafter referred to as phPSA) was kindly supplied by Jeffrey A Medin, Division of Experimental Therapeutics, Ontario Cancer Institute, Toronto, Canada [in vivo vaccination, plasmid DNA was prepared using an Endotoxin free mega kit . Required plasmids were confirmed by enzyme digest and running on 1% agarose gel . Group of mice were also treated with firefly luciferase plasmid (pCMV- luc). The firefly luciferase gene under the control of the CMV promoter was provided by Plasmid Factory GmbH . Plasmid concentrations were determined with the aid of the Nano Drop 1000 Spectrophotometer .The human PSA . The clone was then isolated and purified following three rounds of single cell dilution and designated TRAMPC1/hPSA. The expression of hPSA in TRAMPC1/hPSA was analysed by isolation of RNA and reverse transcription polymerase chain reaction (RT-PCR). For RT-PCR, first strand cDNA was synthesised using omniscript reverse transcription kit . The hPSA cDNA was amplified by PCR, using Pwo polymerase , with hPSA foreword primer (5'-GCAGCATTGA ACCAGAGGAG-3') and hPSA reverse primer (5'-CGATGGTGTC CTTGATCCAC-3'). PCR reaction conditions included 15 min of initial denaturation at 95°C followed by 32 cycles of 1 min at 94°C, 1 min at 57°C, 1 min at 72°C. The wild-type and transfected TRAMPC1 cells were grown in culture at 37°C as reported previously [The murine recycled prostate cancer cell line TRAMPC1 was kindly provided by RP Ciavarra  of Easteeviously .Male C57 BL/6 or MF1-nu/nu mice, 6 - 8 weeks old were used in the study. The mice were obtained from Harlan Laboratories . The animal ethics committee of University College Cork approved all experiments. Mice were kept at a constant room temperature (22°C) with a natural day/night light cycle in a conventional animal colony. Standard laboratory food and water were provided. All mice were maintained in a pathogen free animal facility for at least 2 weeks before the experiments. Subcutaneous (s.c.) tumour inoculation and the tumour growth measurements were recorded (on average every 2 days) as reported previously . A mouseMale C57 BL/6 mice were randomly divided into three groups; phPSA, empty vector and untreated. Mice were anaesthetised during all treatments by intra-peritoneal (i.p.) administration of 200 μg xylazine and 2 mg ketamine. For vaccine delivery, a custom-designed applicator  with two needle electrode (4 mm apart) was used. Both needles were placed through the skin central to the quadriceps muscle. The muscle was injected between electrode needles with 50 μg plasmid DNA in 50 μl sterile phosphate buffer saline (PBS). After 80 s, square-wave pulses  were administered in sequence using a custom-designed pulse generator (Cliniporator). The untreated group did not receive EP. To determine the optimum vaccination protocol, three different regimens of vaccination were tested  by the muscles cells post EP mediated vaccination was demonstrated by RT-PCR of the transfected muscles. After 72 hours of the vaccine delivery, mice were culled by cervical dislocation and quadriceps muscle excised. The muscle tissues were homogenised in TRI Reagent®  to isolate total RNA. DNase treated total RNA from the transfected muscle was subjected to RT-PCR amplification with hPSA specific primers at same conditions (as described earlier).Specific gene expression . Any re-growth of the tumours was observed and growth kinetics recorded.The phPSA induced activation of the immune system and production of interferon gamma (IFNγ), a prototype Th1 cytokine, was tested in vaccinated and naive mice as previously reported . An indiin vitro cytotoxicity assays, splenocytes were isolated from the phPSA treated and naive mice. CTL activity against the TRAMPC1/hPSA cells was analysed using same protocols reported previously [6) in a proportion of 50:1 in serum- free Dulbecco's Modified Eagle's Medium . Tumour devel-opment after inoculation was monitored on alternate days.For eviously ,27. Resueviously . SplenocGACGTTCCTGACGTT (CpG motifs are under-lined) with the backbone phosphorothioate stabilised. The oligo CpG was synthesised by MWG , reconstituted in sterile pyrogen free water, and diluted in PBS for in vivo injection. Three days after each application of EP driven vaccination (regimen 3), mice were injected at same site with synthetic oligo CpG (25 μg/injection). Mice in the control groups were injected either with DNA vaccine or with oligo CpG.CpG oligodeoxynucleotides 1826, chosen according to published data ,30, had The primary outcome variable of the statistical analyses was the tumour volume in each mouse measured at each time point. The principal explanatory variables were the different treatment groups. Tumour volume was analysed as continuous. Treatment groups were analysed as categorical variables. At each time point, a two-sampled t-test was used to compare mean tumour volume within each treatment group. One-way ANOVA was used to compare mean time of tumour appearance in various groups. Animal survival was represented by Kaplan Meyer survival curves. A p value < 0.05 was interpreted as a significant difference. Microsoft Excel 10.0 (Microsoft) was used to manage and analyse data.6/mouse). Nude mice developed tumours earlier than C57 BL/6, which grew more rapidly, resulting in decreased survival of the nude mice (data not shown). These results suggested that the presence of intact immunity in C57 BL/6 mice has some inhibitory effects on the growth of TRAMPC1 tumours, indicating that the TRAMPC1 tumour can be targeted using immune base therapies.To establish the growth and effects of the naive immune reactivity against the recycled TRAMPC1 cells, s.c. tumour inoculation of the TRAMPC1 was performed in both immunocompetent (C57 BL/6) and athymic nude (MF1-nu/nu) male mice with same tumourigenic dose (5 × 10In vivo growth of the wild TRAMPC1 and TRAMPC1/hPSA tumours in C57 BL/6 was comparable (data not shown). This showed that the presence of the human antigen (hPSA) in the TRAMPC1 did not cause significant effects on in vivo tumour growth, validating the suitability of the TRAMPC1/hPSA model.In vivo luciferase activity was demonstrated after EP mediated transfection. The successful transfection of the quadriceps muscle group is shown in a representative image . Additionally the tumour growth was much slower in the immunised group than in untreated (p = 0.02). Although the immunisation resulted in slower tumour growth (compared to both control groups), the difference in growth rate between phPSA and empty vector group was not statistically significant (p = 0.06). The mean survival in immunised mice was 55.6 days, 45 days in empty vector and 42.5 days in untreated mice (p < 0.05) Figure .Regimen 3 was found to be the most effective strategy resulting in delay in time of tumour appearance, retarded tumour growth, and prolonged survival of the tumour bearing mice. The mean time of the tumour appearance was 32.8 days in immunised group (p < 0.01). The tumour growth was also much slower as compared to both groups . These effects were translated into prolonged survival with mean survival after tumour inoculation in phPSA immunised group was 67.5 days, 45 days in empty vector and 40 days in untreated group (p < 0.01) Figure . An overProduction of anti hPSA antibodies in mice serum was determined at various time points after last vaccination. Higher levels of anti-hPSA antibodies were observed at all study time points in regimen 3 and these levels remained persistently higher for up to 12 weeks after last vaccination. Regimen 1 also resulted in production of the anti-hPSA antibodies, but a drop in the level was observed after 4 weeks Figure . After 8Histological analysis (H & E) of the tumours from the immunised mice showed abundant lymphocyte infiltration (data not shown). Immunisation with phPSA also resulted in higher production of IFNγ, indicative of Th1 immune activation. On comparison of the different vaccination protocols, variable amounts of the IFNγ was recorded in the treated mice. However, the levels in regimen 3 were much higher than in other protocols. Mean value of the IFNγ in the regimen 3 was 163.3 pg/ml, while 100.33 pg/ml and 78.33 pg/ml in the regimen 2 and 1 respectively Figure . The regIn vitro cytotoxicity was determined in stimulated splenocytes in all three regimens. Regimen 3 resulted in higher cytotoxicity (75%) than regimen 2 (60%) and regimen 1 (50%)  with longest survival were harvested, mixed with TRAMPC1/hPSA and subsequently inoculated s.c. in groups of C57 BL/6 mice. Fifty percent of the mice receiving mixture of splenocytes from phPSA immunised group failed to develop tumours and importantly the tumour growth in tumour developing mice was significantly retarded (p < 0.01)  Figure . In vivo) Figure . These e) Figure .hPSA).After tumour rechallenge with TRAMPC1/hPSA, only 33% mice developed tumours with previous neo-adjuvant phPSA vaccination Figure . AdditioUse of a synthetic oligo CpG was examined aimed at promoting Th1 type immune responses, to augment potency of the phPSA vaccine. After four vaccines and adjuvant doses of the oligo CpG, 6 out of 11 mice remained tumour free for more than 100 days (cured). Hence, the adjuvant effects of the synthetic oligo CpG resulted in the complete tumour protection (relative risk reduction of 0.45%) as compared with single therapy alone. Time of tumour appearance was also prolonged in combined therapy groups Figure . No signin vitro/in vivo cytotoxicity respectively. These immune responses were antigen specific as in our tumour rechallenge experiments, tumour protection was only observed in mice challenged with transfected cells. In this study, all three tested regimens provided variable effects on tumour growth but repeated vaccination four times on weekly interval resulted more effective immune responses. All mice developed tumours indicating that these regimens did not provide complete tumour protection. Nevertheless, low tumour burden and prolonged survival in immunised mice was achieved with phPSA vaccination with all vaccination regimens. Furthermore, on co-administration of an immune adjuvant, synthetic CpG containing oligonnucleotides, complete tumour protection was achieved in 54% of animals. Immune effects of CpG DNA in infection have been well documented. It has been observed that the release of unmethylated CpG DNA (which is unique to prokaryotes) during an infection provides a 'danger signal' to the innate immune system, triggering a protective immune response that improves the ability of the host to eliminate infecting microbes [Electroporation driven immunisation with prostate antigen (hPSA) encoding plasmid resulted in specific broad immune responses with effective tumour containment. For a cancer vaccine, the prevention of tumour progression may be dependent on both humoral and cellular immunity. We have shown that EP mediated DNA delivery is capable of stimulating both arms of the immune system. Both humoral and cell mediated immune responses were observed as indicated by the anti hPSA antibodies production and microbes . This inmicrobes . Togethemicrobes . These emicrobes ,35. The in vivo transfection and antigen production leading to induction of immunity. A DNA vaccine can activate the innate immune responses by the presence of hypomethylated CpG dinucleotide sequences with particular surrounding motifs in the bacterial plasmid backbone [Developments in tumour vaccines are influenced by the substantial success of the various types of vaccines for infectious diseases. The majority of these vaccines for infectious diseases have effective prophylactic roles with limited utility in therapeutic settings. Tumour vaccine studies have clearly shown that vaccines elicit effective responses against early, microscopic tumours, but are ineffective against established, large tumour masses ,37. Thesbackbone . This mabackbone .at al. have optimised intra-dermal EP mediated PSA DNA vaccination and effectively induced PSA-specific T cells [In terms of induction of immunity, it is difficult to generalise about DNA vaccines. Site and procedure of injection have critical influence on the immune activation. Muscle and skin cells are clearly able to act as antigen depots. The skin contains antigen presenting cells (APCs), hence capable of priming the immune system . Roos at T cells . However T cells . The unc T cells ,44. It i T cells . However T cells ,46. Howe+ T cells are critical. The requirements might be different for the induction of humoral immune responses, for which the induced gene expression level might be of greater importance. Muscle is the most commonly targeted tissue for vaccine delivery where gene expression may last in excess of six months. The dominant mechanism for priming of CD8+ T cells by APCs, after DNA vaccination, is still a matter of debate and may vary depending on if DNA is delivered into the muscle or the skin.The effective delivery of the vaccine vector to the host cells is a prime step for achieving immune activation. We used selected parameters of EP as a tool to boost the transfection of the muscle cells . The traet al. [Despite these promising effects, the clinical efficiency of the different immunotherapeutic strategies for the majority of patients with advanced prostate cancer is still limited owing to various immune evasion mechanisms mediated by tumours. One of the major challenges in developing tumour vaccines relates to the fact that as tumours grow, the immune system looses the ability to target tumour cells, because of development of several immune evading strategies. These mechanisms include down-regulation of different components of the MHC class I processing and presentation machinery, generation of antigen loss variants, production of inhibitory cytokines such as transforming growth factor-ß and IL10, and expression of apoptosis-inducing molecules ,51. DNA et al. . They hain vivo EP mediated vaccination is a safe and effective modality for the treatment of prostate cancer and has potential to be use as a neo-adjuvant or adjuvant therapy.In summary, we have evaluated a plasmid DNA vaccine and shown that this phPSA vaccine can generate effective, durable tumour specific immune responses. The four-dose regimen provided optimal tumour protection and this was further enhanced by co-administration of synthetic oligo CpG. Additionally, this The authors declare that they have no competing interests.in vitro experiments and performed statistical analysis. MT and PS participated in design of the study, helped with data interpretation, and drafting the manuscript. GCOS and MT conceived the study, participated in its design, and coordinated and drafted the manuscript. All authors read and approved the final manuscript.SA carried out mice experiments, performed the statistical analysis, interpreted the data, and drafted the manuscript. GC carried out the


An inflammatory reaction is regularly noticed in irradiated tissues following photodynamic therapy (PDT). This observation is potentially associated with leucocyte-mediated tissue damage, which might further contribute to the tumoricidal effect of this therapy. The objective of our study was to investigate the effects of PDT on leucocyte-endothelium interaction in the microvasculature of tumours and normal tissue. Experiments were performed in the dorsal skinfold chamber preparation of Syrian golden hamsters bearing amelanotic melanoma A-Mel-3. The photosensitiser. Photofrin (5 mg kg-1 i.v.) was injected 24 h before laser irradiation . Post-capillary confluent venules (diameter 15-40 microns) of subcutaneous (s.c.) tissue or the amelanotic melanoma A-Mel-3 were observed by intravital microscopy before, 5, 30, 60 and 180 min after laser irradiation and recorded for off-line analysis. Before treatment, the number of adherent leucocytes in tumour vessels was only 22% of the number observed in vessels of s.c. tissue (P < 0.01). The maximum increase in adhering leucocytes was observed in post-capillary venules of s.c. tissue 1 h after PDT (P < 0.01). In contrast, enhanced leucocyte-endothelium interaction was missing in tumour vessels and in control groups. These results indicate that the tumour destruction observed after PDT is not mediated by leucocyte-endothelium interaction in the tumour. Induction of leucocyte adhesion in the PDT-treated normal tissue suggests a contribution to the peritumoral inflammatory response. Different maturational status or biochemical properties of tumour microvascular endothelium may explain the lack of leucocyte adherence upon PDT.


For tumours to grow they must acquire an adequate blood supply, and the use of drugs to inhibit tumour vascularization is one promising approach to anti-cancer therapy. Clear information is therefore required on the vascular architecture of human tumours and animal tumour models used for testing anti-angiogenic therapies. Many previous studies on animal tumour models have shown that carcinomas are least vascular in their centres and that host tissues become more vascular with proximity to the tumour. However, we have previously found that many human colorectal carcinomas do not show this pattern. The present study on human oral squamous cell carcinomas (SCCs) again reveals significant differences. Paraffin sections from 24 SCCs were immunostained using the QBEnd-10 monoclonal antibody to demonstrate blood vessels, and these were quantified by interactive morphometry using a Kontron Videoplan system. In most carcinomas, viable tumour tissue was no less vascular in the tumour centre than in the tumour periphery. Although tumours are known to release angiogenic factors, viable tumour tissue was less vascular than adjacent host tissues. However, the tumour stroma, by itself, was more vascular than adjacent host tissues. Host tissue adjacent to tumour showed no obvious increase in vascular density with increasing proximity to the tumour edge, which suggests that tumour-released angiogenic factors are only effective over a short distance.


Gradient-recalled echo magnetic resonance imaging (GRE MRI), which gives information on blood flow and oxygenation changes , was used to observe the responses of six rodent tumour models to carbogen breathing. In one transplanted rat tumour, the Morris hepatoma 9618a, and a chemically induced rat tumour, the MNU-induced mammary adenocarcinoma, there were marked image intensity increases, similar to those previously observed in the rat GH3 prolactinoma. In contrast, the rat Walker carcinosarcoma showed no response. In two mouse tumours, the RIF-1 fibrosarcoma and the human xenograft HT29, carbogen breathing induced a transient fall in signal intensity that reversed spontaneously within a few minutes. The rat GH3 prolactinoma was xenografted into nude mice, and an increase in image intensity was found in response to carbogen, suggesting that any effects that carbogen may have had on the host were not significant determinants of the tumour response. The increases in GRE image intensity of the MNU, H9618a and GH3 tumours during carbogen breathing are consistent with increases in tumour oxygenation and blood flow, whereas the responses of the RIF-1 and HT29 tumours may be the result of a transient steal effect followed by homeostatic correction.


We extended these studies to cardiac RyR2 reconstituted into planar lipid bilayers. We found that protamine (0.02–20 µg/ml) added to the cytosolic surface of fully activated RyR2 affected channel activity in a voltage-dependent manner. At membrane voltage  = 0 mV, protamine induced conductance transitions to several intermediate states (substates) as well as full block of RyR2. At Vm>10 mV, the substate with the highest level of conductance was predominant. Increasing Vm from 0 to +80 mV, decreased the number of transitions and residence of the channel in this substate. The drop in current amplitude (full opening to substate) had the same magnitude at 0 and +80 mV despite the ∼3-fold increase in amplitude of the full opening. This is more similar to rectification of channel conductance induced by other polycations than to the action of selective conductance modifiers . A distinctive effect of protamine (which might be shared with polylysines and histones but not with non-peptidic polycations) is the activation of RyR2 in the presence of nanomolar cytosolic Ca2+ and millimolar Mg2+ levels. Our results suggest that RyRs would be subject to dual modulation (activation and block) by polycationic domains of neighboring proteins via electrostatic interactions. Understanding these interactions could be important as such anomalies may be associated with the increased RyR2-mediated Ca2+ leak observed in cardiac diseases.It has been reported that protamine (>10 µg/ml) blocks single skeletal RyR1 channels and inhibits RyR1-mediated Ca Phospholipids were obtained from Avanti . Ryanodine was from Calbiochem . Imperatoxin A (IpTxA) was from Alomone Labs . Ryanodol was obtained from hydrolyzed ryanodine as previously described CaClet al.All procedures with animals were designed to minimize pain and suffering and conformed to the guidelines of the National Institutes of Health. The committee on the Use and Care of Laboratory Animals of Southern Illinois University School of Medicine reviewed and approved the protocols for animal use. Sarcoplasmic reticulum (SR) microsomes were obtained from pig heart ventricle using heart homogenization and ultracentrifugation steps that follow the procedures published by Chamberlain trans compartment was filled with HEPES-Ca2+ solution containing HEPES 250 mM and Ca(OH)2 53 mM, pH 7.4. The trans compartment was clamped at 0 mV using an Axopatch 200B patch-clamp amplifier . The cis compartment (ground) was filled with HEPES-Tris solution containing HEPES 250 mM and TrisOH 118 mM, pH 7.4. Bilayers of a 5∶4∶1 mixture of bovine brain phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine (45–50 mg/ml in decane) were painted onto the holes of teflon septa from the cis side. Sarcoplasmic reticulum microsomes (5–15 µg) were then added to the cis solution followed by 500–1000 mM CsCl and 1 mM CaCl2 to promote vesicle fusion. After RyR currents (or Cl− currents >100 pA at 0 mV) were observed, the cis chamber was perfused with HEPES-TRIS solution for 5 min at 4 ml/min. A mixture of BAPTA and dibromo-BAPTA was used to buffer free [Ca2+] on the cytosolic surface of the channel ([Ca2+]cyt) trans - cis) and response to diagnostic ligands . RyR channel currents are depicted as positive (upward deflections of the current) in figures and reflect cation flux from the trans  to the cis (cytosolic) compartment. Membrane voltages always represent the difference between trans - cis compartments (in mV).Reconstitution of RyR2 in planar lipid bilayers, was performed as previously described o), which were determined by half-amplitude threshold analysis of single-channel recordings as done before Channel currents were first filtered through the Axopatch 200B low-pass Bessel filter at 2 kHz, digitized at 20 kHz with an analog to digital converter  and stored on DVD. Recordings were analyzed using pClamp9 software (Axon Instruments). Analysis with this program included open times, closed times and open probabilities .Frame by frame analysis (100 ms/frame) was performed using pClamp9 on each 4-min recording. In most of our recordings, we were able to distinguish different levels of current that included the baseline, full openings and substates induced by protamine, ryanodol or imperatoxin A. Events, at different levels of current lasting more than 3 ms, were manually selected. The parameters (mean amplitude and duration) of each event were collected and averaged. The probability of substate occurrence, PAll-points current-amplitude histograms (band width = 0.01 pA) were obtained from each 4-min recording. Histograms were normalized so that total histogram area = 1. In most of our experiments, we were able to detect peaks (components) corresponding to the baseline, full opening and substate levels. Each component was fitted with a Gaussian function using the Levenberg-Marquardt method. When the fitting is good, the fitted area of each component can be used as an estimation of its probability.substate were only estimated using frame by frame analysis. It is important to point out that in previous studies o values calculated using different methods).In some cases, the small signal-to-noise ratio precluded the unequivocal resolution of all individual components from the amplitude distribution histograms. In these cases, the Pn measurements. Statistical comparisons between groups were performed with Student's t-test of paired differences. Differences were considered statistically significant at P<0.05.Data are shown as means±S.E.M. of 2+ and caffeine) in order to minimize the interference of RyR2 intrinsic gating events (closures) with the expected effect of protamine . However, other studies have suggested that polycationic peptides  could induce SR Ca2+ release under cellular resting conditions o at low (resting) cytosolic Ca2+ levels.In this work, we studied the action of protamine added to the cytosolic surface of cardiac RyR2 reconstituted into planar lipid bilayers. High concentrations of protamine (>20 µg/ml) have been tested on partially active skeletal RyR1 channels 2+ and caffeine, increasing concentrations of protamine induced sub-conductance states (substates) of several current levels (indicated by dashed lines). With 0.02 µg/ml protamine, infrequent transitions to a substate of ∼50% of the full open conductance were observed. With 0.2 µg/ml protamine, the probability of this substate dramatically increased. Addition of protamine to reach a concentration of 1 µg/ml resulted in the formation of an additional substate of ∼20% the full open conductance. Under these conditions, the probability of full openings decreased to virtually zero and the channel fluctuated between the two substates. With 2 µg/ml protamine, the substate with the smaller conductance became predominant. It is possible that even smaller substates arise at high concentrations of protamine, but they cannot be unequivocally resolved from the baseline current noise. As previously reported for RyR1 As shown in 2+ and caffeine prior to protamine addition.At 0 mV, relative is the ratio of open probabilities (presence of protamine/absence of protamine) measured at each voltage and KPro is the protamine concentration at which block is half-maximal at 0 mV. The “equivalent valence” of the blocker (dz) is influenced by z = 22  and d . This term includes binding site location within the membrane field, Ca2+ flow – protamine interactions and the possibility of multi-sites. Fitting of Woodhull equation to our data render a KPro = 50±23 ng/ml and d = 0.10±0.03. According to the Woodhull model, a d ∼0.1 would indicate that protamine interacts with a RyR2 region that “weakly senses the field”. This resembles previously reported observations for Imperatoxin A (IpTxA) As previously done A)  is an agent that has similarities to the II-III loop of DHPR A-induced substates. We defined “s” as small substate that, in the absence of protamine, has ∼30% of the full opening current amplitude. In the presence of protamine, s is defined as the “sub-substate” and its amplitude is ∼30% of the protamine-induced highest conductance substate. As shown in A and/or protamine). A alone induced frequent long-lived substates. The effect of 1 µg/ml protamine alone is shown in A was tested in two sets of experiments, switching the order in which these agents were applied. The result was always the same regardless of which agent was added in the first place: the channel spent most of the time in the protamine-induced substate with infrequent transitions to sub-substates  was not significantly different in absence versus presence of IpTxA (versus filled triangles). Thus, IpTxA has little or no effect on RyR2 behavior when protamine is present.The peptide imperatoxin A (IpTxubstates . In thistates (A  and a feRyanodol)  . As expeyanodol) , and PRyl values .o in the presence of 100 nM cytosolic Ca2+ as this condition better mimics the channel environment in a resting cell. In the control, at Vm = 20 mV, a few short and infrequent openings were observed (o) was 0.042±0.029 . Addition of 5 mM cytosolic Mg2+ decreased the amplitude of the full open state as well as the substate by ∼15% at 20 mV, which is expected from the decrease in driving force for divalent flux 2+ did not affect protamine induced activation . Notice that in the absence of protamine, at low cytosolic Ca2+ and high Mg2+, RyR2 channels had Po∼0, even when exposed to 10 mM caffeine 2+ flux as it was also observed after replacing lumenal Ca2+ with Ba2+ (not shown), a divalent cation that does not activate RyR2 We tested the effect of protamine on RyR2 with low Pobserved  and the 42±0.029 . Upon ad42±0.029  reaching46±0.013 . As expe46±0.013  and chan09±0.006 . Protami = 40 mV . The act2+. Notice that when the RyR2 activates, it preferentially switches from the closed state to the protamine-induced substate rather than to the full open state (m) increases. This is reminiscent of the rectification of some K+ channels observed in presence of polyamines. Furthermore, protamine activated channels at low resting cytosolic Ca2+ levels (25–100 nM) even in the absence of lumenal Ca2+ or in the presence of high cytosolic Mg2+ levels, suggesting that protamine-induced RyR2 activation mainly results from electrostatic interactions and does not seem to require cytosolic or luminal Ca2+ binding to the respective activating sites.Protamine did not simply block RyR2 in an all-or-none fashion. Rather, it affected the RyR2 channel conduction pathway by producing substates and full block in a voltage-dependent and dose-dependent manner. At low concentrations, protamine induced a drop in the current amplitude to a large conductance substate. The ratio between this amplitude drop and the amplitude of the full opening decreases as the positive holding voltage (Vm = 0 mV) are consistent with previous findings in single RyR1 channels m = 0 mV would lead to a decrease in current flow through the channels, which is in agreement with previous reports were protamine was found to inhibit caffeine-induced RyR1-mediated Ca2+ release from skeletal muscle SR microsomes 2+ release from resting RyR1 and to induce block of drug-activated channels 2+ release in cardiac microsomes regardless of the presence/absence of Mg2+In this work, protamine added to the cytosolic surface of RyR2 had a complex action as it affected RyR2 channel conduction (multiple substates/block) and gating (activation). Our observation of full block of RyR2 induced by high levels of protamine  2+ fluxes increases, it could better counteract an electrostatic screening phenomenom produced by bound protamine and/or change the orientation of protamine putative coiled-coil structure in a rigid vestibule The nature and functional implication of substates in RyRs is not known  and other positively charged “ball peptides” affected the action of ryanodol in a cooperative fashion A were shown to bind to different regions in the RyR molecule Protamine was effective at inducing substates in ryanodol-modified channels, suggesting that the binding sites for protamine and for ryanoids do not overlap. The ryanodol-protamine interactions seem to be cooperative since protamine increased the probability of ryanodol-induced substates. This result resembles previously reported findings, where IpTxIn summary, protamine affects RyR2 channel conduction (substates and block) and the binding affinity of agents that interact with vestibular regions of RyR2. This could be the result of allosterically-induced flexible conformational changes, although steric interference cannot be ruled out. This effect of protamine on conduction does not appear to be highly specific as similar effects were found for other polycationic molecules 2+ levels below those found in resting cells . Regarding the steps involved in protamine-induced activation, our results show that there is a preferential sequence of events. The channels first open to reach the protamine substate level and then they fluctuate between the full open sate and the substate. We also observed that the probability to close is higher when the channel is fully open than during substates. Like protamine, IpTxA- and ryanoids-induced substates rarely transition to the closed state A and ryanoids do not activate RyR2 under resting Ca2+ conditions Protamine (1 µg/ml) activated RyR2 at cytosolic Ca2+, known to compete with Ca2+ at the RyR2 cytosolic binding activating sites 2+ with Ba2+, which would prevent “feed through” activation, as Ba2+ can permeate through the channel but does not activate it 2+- independent within levels found in the cytosol and SR lumen of myocytes.The efficacy of protamine to activate RyR2 was not affected by addition of up to 5 mM Mg2+ dependence of RyR gating is insensitive to carbodiimide titration of negative surface charges located either on the luminal or the cytosolic channel surface 3H]ryanodine binding remained Ca2+ dependent even in presence of very high salt levels (1 M NaCl or KCl) which should greatly affect electrostatic interactions Although RyRs are highly sensitive to the activating action of protamine and polylysine, they are not gated open by a variety of other cationic molecules, including peptides, polyamines and macrolide antibiotics, which only produce substates and/or block 2+ homeostasis.Protamine is clinically utilized to reverse heparin overdose and has been shown to display significant cardiotoxic effects, including decreased cardiac output and electrocardiographic disturbances It is plausible that the strong effect of protamine on channel conduction and gating results from its interaction with the rings of charges in the vestibular region of RyRs 2+ release in skeletal fibers Studies of multichannel behavior indicated that physical RyR-RyR interactions between domains of neighboring channels could shape their coupled gating A and DHPR peptides on sparks suggest they have restricted access to the RyR2 2+ entry is known as an absolute requirement for cardiac EC coupling 2+-entry-independent negative modulation of local events of Ca2+ sparks by specific DHPR agonist and blockers suggests the existence of physical RyR2-DHPR interactions of a still unknown nature that regulate RyR2 function The very limited effects of IpTx2+-independent RyR2 activation and deactivation. The possibility of similar moieties shaping RyR-RyR interactions for functional coupling or DHPR-RyR interactions for triggering and/or terminating SR Ca2+ release opens the field for further investigation.In summary, our studies indicate that protamine, can induce Ca


A crude membrane fraction isolated from mouse tumour cells was treated with various chemicals. The effects on the immunogenicity of the membrane sample were tested in syngeneic mice for tumour protection, using a challenge dose of 10(5) viable tumour cells. Best protection was obtained after immunization of mice with a membrane sample modified with dimethylsulphate. Up to 60% of the animals remained tumour free, and the tumour-bearing animals showed a greatly increased mean survival time. The post-challenge sera contained no detectable amounts of cytotoxic antibodies. The membrane sample isolated from tumour cells which had been modified with dimethylsulphate showed less immunogenicity than the modified cells or the membrane fraction from unmodified cells.


Sclerosing epithelioid fibrosarcoma is a rare but distinct variant of fibrosarcoma that not only presents as a deep-seated mass on the limbs and neck but can also occur adjacent to the fascia or peritoneum, as well as the trunk and spine. We report the case of an intra-abdominal sclerosing epithelioid fibrosarcoma, which to best of the authors' knowledge has not been described previously. The patient discussed here developed lung metastases but is still alive 1-year post-diagnosis.A 29-year-old man presented with a 2-week history of progressive abdominal distension and pain and was found to have marked ascites. A full liver screen was unremarkable with abdominal and chest computed tomography scans only confirming ascites. After a diagnostic laparotomy, biopsies were taken from the greater omentum and peritoneal nodules. Histopathology revealed a malignant tumour composed of sheets and cords of small round cells set in collagenized stroma. After further molecular investigation at the Mayo Clinic, USA, the diagnosis of a high-grade sclerosing epithelioid fibrosarcoma was confirmed.Sclerosing epithelioid fibrosarcoma is an extremely rare tumour, which is often difficult to diagnose and which few pathologists have encountered. This case is particularly unusual because of the intra-abdominal origin of the tumour. Owing to the rarity of sclerosing epithelioid fibrosarcoma, there is no clear evidence regarding the prognosis of such a tumour, although sclerosing epithelioid fibrosarcoma is able to metastasize many years post-presentation. It is important that physicians and pathologists are aware of this unusual tumour. Sclerosing epithelioid fibrosarcoma (SEF) is a rare but distinct variant of fibrosarcoma, which mainly affects young to middle-aged adults of both sexes. Together with low-grade fibromyxoid sarcoma and hyalinizing spindle cell tumour with giant rosettes, SEF belongs to the class of fibrosing fibrosarcomas. It presents as a deep-seated mass on the limbs and neck. Approximately 50% of patients develop local recurrence and/or metastases, but systemic spread is usually delayed for 5 years or more . The maiA 29-year-old man presented with a 2-week history of progressive abdominal distension and pain. On clinical examination, it was found that he had no signs of liver disease or lymphadenopathy, but marked, tense ascites. Although he had had a pansystolic murmur, an echocardiogram revealed only moderate tricuspid regurgitation. A full liver screen was unremarkable, which included a normal ultrasound of the abdomen, with good perihepatic blood flow. Paracentesis demonstrated an exudative nature to the ascites. Reactive mesothelial cells were noted, but not malignant cells. Abdominal and chest computed tomography scans were unremarkable, other than confirming ascites. A diagnostic laparoscopy was initially performed revealing an abnormally thickened and shortened greater omentum and mesentery. This was later converted to a laparotomy as his tumour was buried close to the mesentery of the small bowel, and it was not possible to perform a laparoscopic biopsy safely. Biopsies were taken from the greater omentum and peritoneal nodules.Histopathological analysis revealed a malignant tumour composed of sheets and cords of small, round cells set in abundant collagenous stroma Figure . The celThe immunohistochemical analyses showed positivity for MNF116 Figure  and CAM5Initially, the overall immunochemical profile supported the diagnosis of a primitive neuroectodermal tumour (PNET); however, the morphology was rather unusual. Differential diagnosis included SEF, intra-abdominal desmoplastic round cell tumour and epithelioid synovial sarcoma.in situ hybridisation for Ewing's sarcoma (EWS) locus rearrangement and reverse transcriptase, polymerase chain reaction for EWS-Fli-1 and EWS-ERG (Ewing/PNET), SYT-SSX1 and EWS-WTI .After further molecular investigation at the Mayo Clinic, USA, the diagnosis of a high-grade SEF was confirmed and the differential diagnoses of previously listed tumours were excluded by molecular methods: fluorescence After two courses of palliative chemotherapy, treatment was discontinued at the patient's own request. Subsequently, the patient was admitted to hospital with a large pleural effusion, thought to be metastatic in nature. He remained alive 12 months post-diagnosis.SEF is an uncommon tumour of deep soft tissues, usually affecting adults between 14 and 87 years of age  in 1995,This rare tumour usually affects the lower extremities, limb girdle, trunk and upper extremities in that order ,2,5. OneOwing to the tumour's rarity, the diagnosis of SEF can be difficult. The initial differential diagnosis of this tumour often includes other neoplastic lesions such as carcinoma, lymphoma and other soft-tissue sarcomas. The other sarcomas, which can assume epithelioid morphology, include leiomyosarcoma, malignant peripheral nerve-sheath tumour, epithelial sarcoma, clear cell sarcoma, synovial sarcoma and epithelioid haemangioendothelioma.The diagnosis of SEF is ultimately established histopathologically. The tumour is characterized by an epithelioid phenotype, but in a background of dense hyalinized stroma . FurtherSEF is an extremely rare tumour, which is often difficult to diagnose and which few pathologists have encountered. This case is particularly unusual because of the intra-abdominal origin of this tumour. Owing to the rarity of SEF, there is no clear evidence regarding the prognosis for this tumour, although SEF is able to metastasize many years post-presentation. It is important that physicians and pathologists are aware of this unusual tumour.EWS: Ewing's sarcoma; PNET: primitive neuroectodermal tumour; SEF: sclerosing epithelioid fibrosarcoma.The authors declare that they have no competing interests.PJS was the major contributor in writing the manuscript. BA performed the literature review associated with the case report. JK performed the histological examination of the biopsy and also gave advice on the technical aspects of the case report. BT was the overseeing consultant in charge of the case report. All authors read and approved the final manuscript.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Metastases cause most cancer-related deaths. We investigated the use of hypoxia-selective cytotoxins as adjuvants to radiotherapy in the control of metastatic tumour growth.3) were irradiated with 25 Gy (single fraction) to control primary growth. Equitoxic drug treatments (NLCQ-1 (10 mg kg–1) once daily; RB6145 (75 mg kg–1) and tirapazamine (13 mg kg–1) twice daily) were administered 3–6 days post-radiotherapy when hypoxic cells were evident in lung micrometastases. Mice were culled when 50% of controls exhibited detrimental signs of lung metastases.The NLCQ-1, RB6145 and tirapazamine were assessed against the spontaneously metastasising KHT model. Subcutaneous KHT tumours (250 mmP=0.0002) and RB6145 (60% P=0.02). Semi-quantitative grading of lung disease revealed a significant improvement with all treatments, with NLCQ-1 proving most efficacious ; RB6145, 1 (P<0.001), tirapazamine, 3 (P=0.007)). Positron emission tomography (PET) was evaluated as a non-invasive means of assessing metastatic development. Primary and metastatic KHT tumours showed robust uptake of [18F]fluorodeoxyglucose ([18F]FDG). Metastatic burden discernable by [18F]FDG PET correlated well with macroscopic and histological lung analysis.In total, 95% of control mice presented with lung disease. This was significantly reduced by NLCQ-1 (33% The hypoxia-selective cytotoxin NLCQ-1 controls metastatic disease and may be a successful adjuvant to radiotherapy in the clinical setting. Hypoxia is a common physiological abnormality in solid tumours and is known to be associated with resistance to conventional therapeutics. Further, there is a large body of evidence to suggest that tumour hypoxia is linked to metastatic progression  PET and that lung burden assessed through imaging equated well with alternative assessment methods. Given that metastases are the most common reason for mortality in cancer patients, these findings have marked clinical relevance and support the use of hypoxia-selective chemotherapy in the adjuvant setting.The aim of this study was to ascertain whether hypoxia-selective cytotoxins could be given as adjuvants to radiotherapy to control metastases. This has been analysed using the KHT murine sarcoma established in its syngeneic host (C3H mouse). The KHT model was used as it develops spontaneous lung metastases after primary subcutaneous tumours are controlled with radiotherapy . Recent M glutamine (RPMI complete). The resultant brie was then repeatedly pippeted to disaggregate remaining cell clumps and the suspension plated into a standard culture flask. All in vivo experiments were undertaken using cells established from the same KHT stock tumour after minimal in vitro passaging (<6 passages).KHT cell cultures were established from viably frozen stock tumour pieces. These were rapidly thawed to 37°C, freeze medium removed and the tumour piece cross-chopped in a small volume of RPMI medium supplemented with 10% foetal calf serum and 2 m2, humidified). The MTT  was then added to a final concentration of 5 mg ml–1 for 4 h. Medium was then removed and the cells lysed in dimethylsulphoxide. The optical density (OD) of the formazan product was read at 540 nm using a 96-well plate reader .KHT cells were seeded at 500 cells per well into 96-well plates. Cells were left 2–3 h to attach. NLCQ-1 was prepared at a range of concentrations by serial dilution and added to the cells for 3 h. For anoxic exposure, cell seeding and drug preparation were undertaken in an anoxic chamber  using plastics that had been held within the anoxic chamber for at least 3 days and medium that had similarly been primed to the hypoxic condition. The drug-containing medium was then replaced with fresh culture medium. Cells were cultured for 4 days under standard conditions . Tumour volume measurements were initiated once a palpable tumour formed. Tumour localised radiotherapy (25 Gy) was administered under ambient conditions to non-anaesthetised mice-bearing tumours of 200–300 mm3 in size that were restrained in lead shielded containers. Treatment with bioreductive agents was initiated 72 h after radiotherapy in combination studies. Drugs were all given by intra-peritoneal (IP) injection in fractionated protocols over a 4-day period. Doses were as follows: NLCQ-1, 15 mg kg−1; RB6145, 75 mg kg−1; tirapazamine, 13 mg kg−1. The NLCQ-1 was given once daily and RB6145 and tirapazamine twice daily at 12 h intervals. These were equitoxic dosing regimens for the three compounds. Control mice received 0.9% saline. For metastasis experiments, external evidence of lung metastases in 50% of control mice (weight loss and/or laboured breathing) indicated, from experience, that the experimental end point had been reached. In addition, subsets of tumour-bearing mice were killed 1, 3 and 5 days post-radiotherapy or 16 h after the completion of the treatment with bioreductive drugs. In all treatment groups, pimonidazole  was given 2 h before killing. Lungs and residual primary tumours were rapidly excised and either snap frozen in liquid nitrogen or formalin fixed for histological analysis. As an alternative method for generating lung ‘metastases’, KHT cells were administered by tail-vein injection (5 × 104 cells in 0.1 ml per mouse). Fractionated drug treatments commenced 72 h after cell implant. Mouse welfare was monitored at least daily and more frequently when the experimental end point was approached. All procedures were carried out in accordance with the Scientific Procedures Act 1986 and UKCCCR Guidelines 1997 by approved protocols (Home Office Project Licences 40-1770 and 40-2328 held by IJS and 40/3212 held by KJW) following institutional guidelines.The tumour studies analysing spontaneous metastases formation were performed as earlier described in Primary tumour volumes were monitored post-therapy. Local control was documented when there was no evidence of primary tumour at the point of killing. Local treatment failure or evidence of re-growth described either tumours, which showed little response to the local therapy  or tumours, which initially regressed (often to a point where there was no evidence of tumour), but then re-lapsed and were palpable at killing.After treatment, lungs were rapidly excised and fixed in Bouin's solution  to easily visualise tumour deposits. The severity of metastases was assessed using a semi-quantitative scoring system that has been described earlier , pelleted again and then resuspended in RPMI P/S giving a final preparation equating to 0.1 g lung weight 1 ml−1 medium. This suspension was serially diluted and each dilution plated in duplicate into six-well plates. Colony formation was assessed 10 days later.When the potential onset of metastatic disease was apparent in control mice, mice receiving radiotherapy plus/minus NLCQ-1 were culled and lungs were rapidly excised. They were weighed and an assessment of metastatic burden made according to the criteria listed above. The lungs were cross-chopped in a small volume of RPMI complete, supplemented with penicillin and streptomycin (RPMI P/S). Additional medium was added and the lung brie passed through a sterile 35 18F]FDG scanning ∼9 days after cell implant when xenograft size had reached 200–250 mm3. Animals were fasted overnight before scanning, staggered across the group to reflect scanning times. Animals were anaesthetised with 1–2% isoflurane and injected with ∼10 MBq [18F]FDG  intravenously (IV) through the tail vein. They were then scanned on the quad-high-density avalanche chamber (HIDAC) system  with a theoretical resolution of 1 mm  studies were carried out as described above, with animals imaged under anaesthesia for 60 min after injection with 10 MBq [18F]FLT. [18F]FLT was synthesised ‘in house’ using published protocols  expressed as kBq ml–1. The ROI analysis was undertaken using in-house developed software. Further normalisation was performed using the injected dose  and animal weight to give a standardised uptake value (SUV); SUVmax was calculated from the maximum voxel value within the ROI and SUVmean as the average overall voxels. For [18F]FDG, normalised uptake values (NUVs) were calculated by dividing SUVmax or SUVmean from the tumour by that from the brain.Tumour studies for PET were carried out as described above. For the pilot study, animals underwent dynamic ).To complement the studies into effects against metastases that spontaneously arise from subcutaneous KHT tumours, experiments were undertaken to assess the impact of NLCQ-1 and tirapazamine treatment on lung metastases formation after intravenous injection of KHT tumour cells . Initial studies were undertaken to determine the injected cell number required to give metastases over the same time frame as the interval between radiation and morbidity in the spontaneous model. In addition, histology was undertaken to ensure that metastases were present over the course of bioreductive treatment . Neither NLCQ-1 nor tirapazamine caused a significant change in the distribution of metastastic grade observed compared with saline-treated controls (n=4 per group). The KHT model is highly vascularised and control tumours had a vessel density of 86±13 (s.e.) mm−2. The NLCQ-1 reduced this slightly to 67±17 (s.e.) mm−2, but the change was not significant (P=0.48). Concomitant with the reduction in vessel density, irradiated NLCQ-1-treated tumours exhibited an increase in hypoxic fraction compared with saline-treated controls (13±3 vs 9±1 in controls), but again this did not achieve significance (P>0.05).Recent data have suggested that some bioreductive agents may have anti-angiogenic effects  with those determined using the semi-quantitative visual scoring method  showed an average of 165±57 clonogens per mg. Tumour tracer uptake was normalised to that observed in the brain. There was no significant difference in NUVs between treated and untreated primary tumours or metastases. The NUVmax and NUV mean were 1.1±0.3 and 1.0±0.3 for untreated primary tumours and 1.0±0.4 and 0.9±0.2 for treated tumours. Comparative values for metastases were NUVmax 0.9±0.2 for both groups and NUVmean 1.4±0.2 and 1.3±0.3 for untreated and treated, respectively.in vivo (NLCQ-1 (NSC 709257) is the lead bioreductive agent from a rational approach to develop nitroaromatics with weak DNA-intercalating properties that may improve drug biodistribution . Earlierin vivo , 2007. HThe KHT tumour model is a highly aggressive, locally invasive sarcoma that spontaneously metastasises to the lungs. It is almost unique in the reproducibility of the temporal onset of lung metastases, making it an ideal system in which to study anti-metastatic approaches. Our hypothesis in this study was that early stage metastases have a reliance on hypoxia to initiate gene expression changes that precipitate their subsequent growth. In preliminary studies, we were able to show that early stage microscopic KHT lung foci can be hypoxic. Up to day 5 post-radiotherapy of the primary tumour, there was no evidence of vascularisation of the hypoxic micrometastases. On the basis of the presence of a target population in the metastases, bioreductive treatment was given over 4 days starting 3 days after radiotherapy of the primary tumour.vs experimental metastases perhaps supports that NLCQ-1 is indeed targeting an early step in spontaneous metastasis. Further, it may suggest that there are pathophysiologic differences between the two models. This may be associated with potential route of dissemination, given that cells could distribute from a primary tumour through lymphatic or blood vessels, whereas tail-vein injection forces dissemination through the latter route. Interestingly, pathological assessment of lungs from the spontaneous groups suggested lymphatic dissemination (data not shown). This speculation is perhaps supported by the presence of lymph node involvement as identified by [18F]FDG PET.Both RB6145 and tirapazamine improved the local control of the primary tumour, whereas NLCQ-1 had little effect. However, NLCQ-1 was the most effective at controlling metastases. Improved local control could conceivably influence the dissemination of tumour cells from the primary site. However, the fact that NLCQ-1 has little impact on local control suggests an alternative mechanism of action. We would argue that a direct effect against the hypoxic metastases is a strong candidate. In an attempt to remove the potential influence of primary tumour effects, the efficacy of NLCQ-1 and tirapazamine against KHT lung metastases established after tail-vein injection was assessed. Neither agent significantly affected metastatic growth. The apparent differences in efficacy against spontaneous In our earlier studies, tirapazamine given before radiotherapy was able to reduce subsequent metastases formation, whereas RB6145 showed little effect , this pe18F]FDG PET. The KHT model both established subcutaneously and when disseminated to the lung showed excellent uptake of [18F]FDG. In addition, there was a highly significant correlation between the metastatic profile obtained through [18F]FDG PET and that after histological examination of the lungs after killing of the tumour-bearing mice. Importantly, lungs that scored zero also had a low percentage of viable cells as measured by clonogenic assay. In addition, they had either no or single metastases discernible by [18F]FDG PET. This indicates that there is not significant viable cell mass escaping detection with [18F]FDG PET. As was observed in the efficacy studies, NLCQ-1 seemed to reduce the metastases detected by [18F]FDG PET at 20 days post-radiotherapy. Although partial volume effects will lead to a lessening of metastatic SUVs (18F]FDG in primary KHT tumours (18F]fluoroacetate and [18F]fluoromisonidazole in this model. Given that our data also show strong uptake of [18F]FLT in primary tumours, this suggests that this fast-growing KHT model has particular usage in longitudinal PET-based imaging studies to assess tumour response to a range of targeted therapies in an immunocompetent host.Although the KHT is an optimal model in many respects, one issue is that it is not possible to readily establish genetically modified clonal populations from these cells. This negates the possibility of using such approaches as bio-luminescence as non-invasive approaches to monitor disseminated tumour growth. We investigated whether the lung metastases could be resolved using [Overall, we summarise that bioreductive agents such as NLCQ-1 could have potential usage as adjuvants to radiotherapy in the control of metastatic disease and that clinically applicable non-invasive PET imaging can be used to monitor drug efficacy in the KHT model. Earlier studies have shown that bioreductive strategies synergise not only with radiotherapy, but also commonly used chemo- and chemo-radiotherapy in which hypoxia limits the effectiveness of treatment (


I.p. or i.v. administration of Corynebacterium parvum (CP) to MF1 mice induces a generalized inflammatory response, associated with marked hepatosplenomegaly and accompanied by a pronounced granulomatous response in the liver. Injection of the Landschütz ascites carcinoma (LAC) 24 h after CP substantially reduced the intensity of the inflammatory response, and decreased both the frequency and size of the hepatic granulomas, as revealed by morphometric analysis of histological sections. The difference in cellular composition of the granulomas between the experimental groups, as revealed by light microscopy, was further emphasized and characterized by ultrastructural studies. These revealed the predominance of macrophages within the granulomas in tumour-bearing mice, in contrast to the predominance of epithelioid cells in the lesions which developed in mice given CP alone. Our experimental findings show that the inhibitory effect of the growing LAC on granuloma formation in response to CP cannot be ascribed to (a) sequestration of the microorganism within the growing tumour, (b) a nonspecific inflammatory stimulus, (c) diversion and seqestration of mononuclear phagocytes in the growing tumour or (d) the presence of lactate dehydrogenase-elevating virus in either the host or tumour cells. The inhibition of liver granuloma formation is consistent with an effect mediated by soluble, heat-stable tumour-associated factor(s).


Tumour cells at the invasive front of carcinomas have been found to differ substantially from the rest of tumour cells in a variety of human cancers. The present multivariate survival analysis of 94 oral squamous cell carcinomas (OSCCs) revealed that both the argyrophilic nucleolar organizer regions-associated protein (AgNOR) content of invading tumour cells and a multiparametric histopathological tumour front grade were significantly and independently associated with tumour-related death, irrespective of conventional Broders' grade and clinical stage of the tumours. High tumour front scores and AgNOR content at the invasive OSCC front thus seem to reflect increased malignant potential. Proliferative activity, assessed by standardized AgNOR analysis, most probably represents one of the biological features underlying the usefulness of evaluating the invasive tumour front.


When normal rat and mouse embryo cells were treated with a cell free extract of S37 ascites tumour, morphological transformations occurred in both. The transformed cells readily induced lymphosarcoma-type tumours in the mice inoculated when newborn or young adults (8-12 weeks old), but not so readily in rats.Various tests carried out with these cells strongly indicate the presence of an oncogenic and transmissible agent in S37 ascites tumour. This agent appears to be related to mouse sarcoma virus isolated from animals with Moloney leukaemia but differs in only producing characteristic lymphosarcoma-type tumours.


X death time, (2) protamine was more toxic for leukemic than normal mice and (3) the in vitro reaction between Rauscher virus and protamine reduced its infectivity for mice.This study indicated that: (1) i.p. inoculation of protamine into (Rauscher) leukemic mice increased their


We investigated the effects of low-dose eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on the incidence and growth of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in rats fed a high-fat (HF) diet. We also examined the effects of these treatments on the fatty acid composition of tumour and serum. Tumour incidence was significantly decreased by the administration of low-dose EPA and DHA, whereas their inhibitory effects on tumour growth did not reach significance. Serum arachidonic acid (AA) level was decreased by the administration of low-dose EPA and tended to be decreased by the administration of low-dose DHA, whereas tumour AA levels were not changed. The administration of low-dose EPA and DHA may be useful for inhibiting the incidence of breast cancer.


Observations on the in vivo plating of mouse mammary tumour are extended by making counts of tumours at a significantly earlier phase of development than in previously reported work. In the experiments now described, most of the growth of the tumours has been without benefit of stroma. The noteworthy economy of the experimental method is discussed. The persistence of endotoxin's adjuvator effect on such tumour counts is tested in the face of gamma irradiation and cortisone. Cortisone, it is found, offsets endotoxin's adjuvator action; irradiation does not. Antagonism between endotoxin and cortisone, in this system with tumour cells plated in vivo, seems to indicate that endotoxin's enhancing effect depends more on inflammatory than on immunological factors.


In the rat, this tumour produces osteolytic bone deposits and hypercalcaemia, both of which can be prevented by aspirin and indomethacin, whereas soft tissue tumour deposits are unaffected by these drugs. Some human breast tumours cause in vitro osteolysis which may be inhibited by aspirin.Walker carcinosarcoma cells cause


Bacteriochlorin a (BCA) is a second-generation photosensitizer that is effective in tumour destruction upon illumination with light of a wavelength of 760 nm. Tissue penetration by light at this wavelength is greater compared with wavelengths at which commonly used photosensitizers are illuminated, making it possible to treat larger tumours. In a model of experimental liver metastases in rats, we measured lesion sizes after interstitial illumination of tumours at different times after intravenous administration of BCA (10 mg kg(-1) bodyweight), as well as BCA concentrations in liver and tumour tissue. In both, BCA concentrations showed a rapid decline within the first 4 h, followed by a slow decrease over the next 20 h, suggesting biphasic pharmacokinetics. No selective uptake in tumour tissue was observed. A near-linear relationship was found between lesion sizes and liver and tumour BCA concentrations, suggesting that optimal results with photodynamic therapy (PDT) could be obtained by illumination within a short time interval after administration, when tissue concentrations are highest. No severe liver toxicity was observed as indicated by serum ALAT levels. However, in all tumours evaluated, islands of vital-looking cells were present leading to tumour regrowth within 35 days. In view of the obtained lesion diameters of approximately 13 mm after BCA-PDT and the rapid clearance rate of BCA, the concept of a near-infrared absorbing photosensitizer for PDT of liver tumours is a potential interesting strategy.


Pulmonary growths produced by intravenous injection of methycholanthrene-induced sarcoma cells were controlled or suppressed by specific immunostimulation with BCG-sarcoma cell inocula at subcutaneous sites or by intravenous treatment with BCG alone. The response was dependent upon the immunogenicity of the treated tumour so that in comparison, intravenous injection of BCG enhanced rather than suppressed pulmonary growths of transferred cells of several weakly immunogenic tumours including a spontaneously arising sarcoma and an epithelioma as well as a carcinogen-induced mammary carcinoma.


Nuclear magnetic resonance (NMR) non-invasively measures the apparent diffusion coefficient (ADC) of water, which is sensitive to the biophysical characteristics of tissue. Because anti-cancer treatment alters tumour pathophysiology, tumour ADC may be altered by treatment. In order to test this hypothesis, ADC was measured in s.c. implanted murine RIF-1 tumours before and up to 9 days after treatment with cyclophosphamide. A dose-dependent, reversible increase in tumour ADC was observed after cyclophosphamide treatment, which is consistent with an increase in the fraction of interstitial water due to treatment-induced cell death. Because tumour water ADC is increased substantially at a time when there is no change in tumour volume for a dose which produces minimal cell kill, its measurement could provide a novel means for early detection of response to anti-cancer therapy. If the changes in ADC observed in the present study are evident for commonly used anti-cancer therapies in different tumour types and specific to a therapeutic response, the approach could be broadly applicable as a response predictor since magnetic resonance imaging can be used to measure ADC in human tumours.


Serum inhibition of autochthonous lymphocyte cytotoxicity for tumour cells has been studied in 112 cases of colonic carcinoma. Addition of patient's serum to the lymphocyte tumour cell reaction mixture resulted in decreased cytotoxic reactivity of lymphocytes from 8 of 39 cytotoxic positive cases. It was also shown that sera could inhibit if separately preincubated with the lymphocytes (4 cases) or the target cells (2 cases). A tumour antigen preparation inhibited only when incubated with the lymphocytes. Inhibition by serum or antigen appeared to be specific for colon carcinoma. Four cases were specially studied to determine the mode of lymphocyte killing of tumour cells: in 3 it was mediated largely if not entirely by T lymphocytes, and in the fourth by both T and non-T cells. The findings support the view that T lymphocytes lose their anti-tumour reactivity in vivo in the presence of circulating antigen or antigen-antibody complexes such as would occur with progressive tumour growth.


Hepatic parenchymal vasoconstriction increases cytotoxic drug uptake into hepatic metastases by increasing the tumour to liver blood flow ratio. Prolonged infusion of the vasoconstrictor vasopressin does not result in sustained vasoconstriction, and this may limit the benefit of vasopressin in infusional chemotherapy. We have assessed whether loss of vasopressin-induced vasoconstriction is mediated by nitric oxide. Hepatic and tumour blood flow were continuously monitored, in an animal hepatic tumour model, by laser Doppler flowmetry. The response to regionally infused vasopressin and the nitric oxide inhibitor N-nitro-L-arginine methyl ester (L-NAME) were assessed over a 30 min infusion period. The vasopressin-induced vasoconstrictor effect diminished after 15 min despite continued infusion. Vasoconstriction was significantly prolonged when L-NAME was infused in addition to vasopressin. The increase in tumour to normal blood flow ratio was greater over the infusion period when L-NAME was co-administered with vasopressin. Our results suggest that the loss of vasopressin-induced vasoconstriction seen in liver parenchyma after regional infusion is prevented by the nitric oxide synthase inhibitor L-name and may be mediated by nitric oxide.


The growth of a CBA mammary adenocarcinoma has been studied following transplantation to syngeneic and allogeneic recipients, with particular reference to the susceptibilities of the primary and secondary responses elicited by the tumour allografts, to impairment by whole-body X-irradiation and by treatment with rabbit-anti-mouse lymphocyte serum. In syngeneic recipients, the diameter of tumour implants increases linearly with time and there is no difference in the growth curves in females and in males. Later tumour generations grow faster than earlier generations. In allogeneic recipients, there is a relationship between the tumour diameter on day 21 (T) and the dose of X-irradiation (D) administered before implantation:T = 0.028 D - 9.17for early tumour generations (SMT4) but this is obscured for later generations (SMT21). The primary response to tumour allografts was radiosensitive whereas the secondary response was radioresistant. This radioresistance of the secondary response persisted for at least 5 months after primary sensitization. Unlike whole-body X-irradiation, treatment with rabbit-anti-mouse lymphocyte serum suppresses both the primary and secondary responses to tumour allografts. The possibility is considered that after exposure to antigenic stimulation, an immunologically reactive cell population is formed which is radioresistant but sensitive to ALS, unlike the precursor cells from which this population is derived, which are radiosensitive and sensitive to ALS.


Congenital Epulis, also known as Neumann's tumour, is a rare congenital growth affecting the gingival mucosa of neonates. It is benign condition, seen more frequently in females, with multiple Epuli occurring in only 10% of cases. The cause and origin of Congenital Epulis remains unclear. In this article we present a case report of an otherwise healthy female neonate with two Congenital Epuli arising from the upper and lower gingival margin, which were successfully treated with surgical excision. We also present a review of the literature and an estimation of the incidence of Congenital Epulis based on our institutions figures, of 0.0006% . Congenital Epulis  is a rare benign congenital growth of the newborn. It was first described in 1871 by Neumann . There iAn otherwise healthy 1-day-old girl was referred to a large teaching hospital in Cardiff for diagnosis and treatment of two large masses protruding from her mouth. The baby had normal antenatal scans at weeks 12 and 20, and pregnancy had been unremarkable, other than mother being Group B Streptococcus positive from a high vaginal swab. Mother was fit and well gravida 2 para 1, with no drug history or family history of note. Baby was born at term plus eight days weighing 3.85 kg, pink and breathing spontaneously (Apgar: 9-10).On examination there were two fleshy, pedunculated masses arising from the upper and lower alveolar ridges measuring 4 × 3 × 3 cm just to the right of the midline. There were no respiratory difficulties. A nasogastric tube was passed due to concerns over feeding. At that time a differential diagnosis of Congenital Epulis, Haemangiomas, and Teramomas was made.She was booked for excision of these masses under general anaesthesia . Both maThe two masses were fixed and examined histologically Figures and 4. ThCongenital Epulis is a rare tumour of the neonate. Zucker and Buenecha  found onα, whereas Congenital Epulis does not.Epulis is a Greek term literally meaning “of the gums” and is used to describe a wide variety of gum lesions, regardless of their pathological origin. Histologically, Congenial Epulis shows remarkable similarity with the more common Granular Cell Tumours (GCTs) , 11. TheThe precise origin of Congenital Epulis remains unclear. CGTs are considered to arise from Schwann Cells, and hence show strong reactivity to S-100 protein . VariousNo estimation of incidence of Congenital Epulis has been made to date, to the best of our knowledge. One centre in the USA saw only two cases over the period of 21 years . In Univ


Two 3-methylcholanthrene and a spontaneous BALB/c fibrosarcoma were examined for tumour-associated antigens (TAA) by in vivo and in vitro induction of tumour-immune responses. When BALB/c mice were immunized to these fibrosarcomas by surgical tumour removal, cross-reacting tumour-associated transplantation antigens (TATA) were detected on all 3 tumours. Cytotoxic effector cells (CL) were then induced in vitro by co-culture of BALB/c spleen cells with the spontaneous, or one of the carcinogen-induced fibrosarcomas. These CL were shown to be cytotoxic T cells (Tc) and to be directed against cross-reacting TAA on all 3 tumours, by two in vitro 51Cr-release assay systems, direct 51Cr-release cytotoxicity and cellular competitive inhibition of 51Cr release. Further studies demonstrated that the fibrosarcoma TAA involved in in vitro induction of Tc were not present on normal adult or foetal tissues. A secondary cytotoxic response was also detected in vitro when spleen cells from mice immunized to a carcinogen-induced fibrosarcoma were tested. The patterns of cross-reactivity detected by the in vivo and primary in vitro tumour-immune responses suggested that the TAA detected in vivo (TATA) were not identical to the TAA detected in vitro.


Cancer Res57: 156–161). If sigma-2 receptors were a biomarker of tumour cell proliferation, they would be amenable to detection by non-invasive imaging procedures, thus eliminating many of the problems associated with the flow cytometric measures of tumour cell proliferation presently used in the clinic. To be a good biomarker of tumour cell proliferation, the expression of sigma-2 receptors must be essentially independent of many of the biological, physiological, and/or environmental properties that are found in solid tumours. In the investigation reported here, the mouse mammary adenocarcinoma lines, 66 (diploid) and 67 (aneuploid), 9L rat brain tumour cells, and MCF-7 human breast tumour cells were used to study the extent and kinetics of expression of sigma-2 receptors in proliferative (P) and quiescent (Q) tumour cells as a function of species, cell type, ploidy, pH, nutrient depletion, metabolic state, recruitment from the Q-cell compartment to the P-cell compartment, and treatment with tamoxifen. In these experiments, the expression of sigma-2 receptors solely reflected the proliferative status of the tumour cells. None of the biological, physiological, or environmental properties that were investigated had a measurable effect on the expression of sigma-2 receptors in these model systems. Consequently, these data suggest that the proliferative status of tumours and normal tissues can be non-invasively assessed using radiolabelled ligands that selectively bind sigma-2 receptors. © 1999 Cancer Research CampaignRecently, we demonstrated that sigma-2 receptors may have the potential to be a biomarker of tumour cell proliferation


The cell population kinetics of the transmissible venereal tumour of the dog was studied at two different stages of tumour growth using the labelled mitoses technique. At the first stage the tumours were growing with a doubling time of about 4 days; at the second stage their growth rate was limited, probably by an immune reaction on the part of the host, to a doubling time greater than 20 days.Labelling of the tumour cells was found to be extremely heterogeneous throughout the tumour. Mitotic figures, however, were present in well labelled as well as in poorly labelled fields, suggesting that thymidine did not reach all regions of the tumour nodules. The data were therefore analysed assuming that the cells in well labelled areas were representative of the total cell population in the neoplasm. The timing of the cell cycle was found to be similar in the rapidly growing tumours and in those growing more slowly. It is concluded that the slowing of growth was due to a considerable increase in the rate of cell loss as a result of the immune reaction.


Generalized lymphosarcomatosis (leukaemia) of non-thymic type occurs in mice bearing 90Sr or 239Pu or 226Ra. Tumours passaged from such mice have been tested for tumour-associated transplantation antigens that could provoke a protective immunity which would be expected if such antigens were determined by virus activated by the irradiation. Sub-threshold doses of living syngeneic tumour, large doses of living allogeneic tumour and large doses of killed syngeneic tumour were without protective effect. This suggests that viruses observed electron micrographically in such tumours are passengers and not causative.


Lymphatic vessel spread is considered a major route for head and neck squamous cell carcinoma metastasis. Formation of new lymphatic vessels could facilitate the process, raising the malignant potential of these tumours. Recent identification of lymphatic markers allows the study of the lymphangiogenesis phenomenon. We searched for molecular events involved in the lymphangiogenic process that could have prognostic value in laryngeal/pharyngeal carcinoma patients.104 paraffin-embedded pharyngeal/laryngeal tumour samples were studied. Immunohistochemical analysis of podoplanin and double immunofluorescence analysis of Ki-67 and D2-40 were performed. Lymph vessel density  and the presence of tumour emboli inside lymphatics were recorded. The proliferative state of endothelial lymphatic cells was evaluated.Lymphatic vessels were detected inside the tumour mass (75%) and in the surrounding tissue (80%); some of them in a proliferative state. Tumour emboli were detected in a high proportion of the cases (45%). Lymphatic vessel density was higher in the pharyngeal cases (p = 0.0029), in greater size (p = 0.039), more advanced stage primary tumours (p = 0.006) and in carcinomas of patients with affected nodes (p = 0.019). The presence of tumour emboli and a high global vessel density were indicators of poor prognosis (recorded as death from tumour) in the laryngeal group , but notably not in the pharyngeal one. Interestingly, high global vessel density showed a negative prognostic value among pathologically staged N0 laryngeal carcinomas (p = 0.03).The lymphangiogenic process correlated with aggressive tumour features , but might play different roles in tumours arising from different anatomic sites.Our results suggest that detection of tumour emboli and assessment of global vessel density using the D2-40 antibody, may be useful in the clinical practice, as predictors of reduced survival among pN0 laryngeal carcinoma patients. Head and neck squamous cell carcinoma (HNSCC) remains a significant cause of morbidity and mortality, afflicting 500.000 new cases worldwide each year. The sinInvasion of cells into the surrounding tissue and the destruction of normal tissue architecture are two hallmarks of malignant tumours. Lymphatic vessels serve as the primary conduit for malignant tumour cell to regional lymph nodes. In receUsing the D2-40 antibody in this study, the presence and density of lymphatic vessels were determined and quantified in laryngeal/pharyngeal carcinomas, and their potential prognostic values were assessed.Paraffin-embedded tissues from 104 patients with pharyngeal or laryngeal squamous cell carcinoma  and antigen retrieval , immunostaining was performed with D2-40 mouse monoclonal antibody against human podoplanin  . Staining was done by using the DakoCytomation Envision Plus peroxidase mouse system. The stained protein was visualized using the DAB solution (Dako), and lightly counterstained with Mayers-haematoxilyn.To ascertain the specificity of the antibody immunoreactivity, a negative control was carried out by exclusion of the primary antibody. In this case, immunolabeling was completely abolished.2.Quantitative analysis of the intratumoural and peritumoural lymphatic vessel density was performed by two independent observers (DGC and MVG) in a blinded fashion. Three fields with the highest lymphatic vascular density were identified , and the vessels were counted (×400 magnification) both within the tumour area  and within an area 500 μm from the tumour border . Lymphatic vessel density (LVD), regardless of the vessel location with respect to the tumour, was also considered. Lymphatic density was defined as the number of lymphatic vessels per mmWe also evaluated invasion of the podoplanin-positive lymphatic vessels by cancer cells. Lymphatic invasion (LI) was considered to be present if at least one tumour cell cluster (a tumour embolus) was clearly visible inside a podoplanin-positive vessel. Cases were classified as LI positive or negative depending on the presence or absence of tumour emboli, respectively.202 and 50% fetal calf serum (Sigma), respectively, and the sections were incubated overnight with the primary antibodies in a humid chamber at 4°C: mouse D2-40  and rabbit anti-ki67 (Abcam ab833). After the incubation with the primary antibodies, sections were incubated for 1 hr at room temperature with Texas red-labelled sheep anti-rabbit immunoglobulin G (IgG) and fluorescein isothiocyanate-conjugated sheep anti-mouse IgG, both diluted at 1:100 (Amersham). Finally, tissue sections were photographed with a confocal-laser scanning microscope .Ten representative cases were selected on the basis of the images of the lymphatic vessels obtained in D2-40 IHC. For double immunostaining, deparaffined and rehydrated sections were rinsed in 0.05 M Tris-buffered saline  containing 0.1% bovine serum albumin and 0.2% Triton X-100. Endogenous peroxidase activity and non-specific binding were blocked with 3% H2 test for categorical variables and t-Student/ANOVA or Mann Whitney/Kruskal-Wallis tests for parametric and non parametric continuous variables, respectively. Survival curves  were calculated using the Kaplan-Meier product limit estimate. Differences between survival times were analyzed by the log-rank method. Multivariate Cox proportional hazards models were used to examine the relative impact of either variables demonstrated to be statistically significant in univariate analysis or those variables likely to have an effect on the outcome. P-values < 0.05  were considered statistically significant. All the statistical analyses were performed with the statistical software SPSS 12.0 .For statistical purposes, clinicopathological features were dichotomized as: pT category: 1-2, 3-4; pN category: N0, N1-3; TNM stage: I-II, III-IV. The molecular data distributed among the different clinical groups of tumours were tested for significance employing the χResults are reported following the REMARK guidelines (REporting recommendations for tumor MARKer prognostic studies).Immunohistochemical detection of podoplanin with D2-40 antibody shows strong specific reactivity with lymphatic endothelial cells, while the endothelial cells of blood vessels show no signal Figure .D2-40-positive lymphatic vessels could be identified between islands of neoplastic epithelium Figure  and at tThe percentages of the cases with positive lymphatic vessel stainings, locations of the positive standings , mean values, and score ranges in the 104 HNSCC, 54 laryngeal and 50 pharyngeal carcinoma samples are summarized in Tables Double immunofluorescence assay was performed with D2-40 and the proliferation marker ki-67 in ten representative cases. Vessels were deemed positive for proliferation when they harboured at least one ki-67 positive cell Figure  and werePodoplanin-positive carcinoma cells could be visualized inside lymph vessels (irrespective of vessel location) in 43% of the cases  Figure . Figure Considering the whole series for analysis, higher mean ILD, PLD and LVD values were significantly associated with greater tumour size, lymph node metastasis, advanced tumour stage and pharyngeal tumour site Table . Tables The normal glottis is a particularly interesting site, since it is a lymph vessel free region. In our series, 13 out of the 29 glottic cases (45%) showed lymphatics within the tumour mass, 3 of which were T1 tumours.vs. 58% in pharyngeal tumours; p = 0.002) and were more frequently observed in tumours of patients with affected lymph nodes (p = 0.001), in greater size tumours (p = 0.006) and in more advanced stage tumours (p = 0.000).Importantly, the presence of tumour emboli (lymphatic invasion) correlated with anatomic site .vs. the rest, attending to the median value of the lymph vessel density in the positive population  were categorized into high vessel density cases on Table . In thison Table  and overon Table  but not A very interesting observation could be made for the 47 HNSCC patients with pN0 tumours : 19 cases showing a high LVD  had a poorer prognosis than the rest (p = 0.005) Figure . Among tNone of these variables reached statistical significance when multivariate Cox analysis was performed.In the present study we investigated the existence of tumour lymphangiogenesis in a cohort of 104 laryngeal/pharyngeal carcinoma patients and evaluated whether ILD, PLD or LVD correlated with the presence of lymph node metastasis; we also addressed the prognostic significance of the above parameters on the disease specific and overall survival of these patients.As previously described, podoplanin was detected in tumour cells  and lympIn reference to the existence of dividing cells among lymphatic endothelial cells, we found evidence of at least one proliferative endothelial lymphatic cell in some tumour sections, although their proportion was low. These results are in line with previous reports that point to the existence of an active lymphangiogenesis process in HNSCC,15.The correlation between ILD and nodal involvement supports the role of intratumoural lymph vessels as a possible route for the spread of tumours to local lymph nodes. Interestingly, tumour emboli  were observed within lymphatics in 43% of tumour samples, a higher frequency than previously reported,15,17,18Lymphatic cell proliferation and tumour emboli formation may be rare events, difficult to detect by examining small sections of archival tissue at a single time point. Despite this, the detection of proliferating lymphatics in carcinoma tissue and of tumour emboli within lymphatics constitutes considerable evidence for the presence of active lymphangiogenesis in this type of malignancy. Additionally, we present evidence of a true lymphangiogenic process in the larynx, since lymphatics were detected in pT1 tumours arising in the glottis, a physiologically lymph vessel free site. This observation would indicate the formation of new vessels in these cases. On the other hand, the presence of lymphatics in the pT2-4 glottic tumours would not necessarily point to this phenomenon, since vessels might also result from their being entrapped as growing tumours reached the vessel rich areas, such as the paraglottic space, supraglottis or subglottis.vs. peritumoural vessel identification when studying single tissue sections taken at a single time point, which might yield a misleading vessel classification, limiting the potential value of these variables. Although both ILD and PLD contribute to tumour spread to lymph nodes, from our experience, LVD counting (regardless of their location with respect to the tumour) is more reproducible and might be a more reliable marker of poor prognosis, especially within the laryngeal subgroup.Controversial results can be found in the literature regarding the specific contribution of intra and peritumoural lymphatic vessels to tumour spread. Functional studies in animal models have shown that intratumoural lymphatic vessels are compressed and nonfunctional. However, some studies on human tumour samples reveal that intratumoural lymphatics are involved in nodal metastasis and that ILD is a significant indicator of poor prognosis,14,17. ORemarkably, although nodal involvement had a negative prognostic value, this prediction could be refined among the pN0 laryngeal tumour population, according to their LVD. Thus, significant differences on survival times were found for cases showing a high LVD (poorer outcome). This observation was also made in the whole series of carcinomas but not in the pharyngeal subgroup, and might be a useful tool in the pN0 laryngeal cancer patients' clinical management.In summary, our results suggest a differential implication of lymphangiogenesis in tumours arising from different anatomical head and neck sites. We propose that LVD assessment and tumour emboli detection using the D2-40 antibody should be considered as potential prognostic tools for laryngeal tumours. This would merit further large-scale validation analyses for the eventual future incorporation of these potential markers into clinical practice.The authors declare that they have no competing interests.DGC carried out the IHC studies and vessel counting and helped to draft the manuscript. JPR participated in its design and contributed to clinical data collection and results interpretation. AA contributed to the pathological assessment of tissue sections. CSN participated in its design and coordination. MVG conceived the study, coordinated and supervised its progression, carried out vessel counting, performed the statistical analysis and wrote the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/416/prepub


Effects of amphotericin B on production of endogenous tumour necrosis factor alpha  and anti-tumour activity in mice was examined. Intravenous administration of Fungizone, an amphotericin B preparation complexed with deoxycholate, augmented the induction of endogenous TNF in response to a second stimulus with intravenous doses of FK23 . This augmentation was observed when more than 1.8 microg of Fungizone was injected intravenously before intravenous dosing of FK23. The time interval between priming injection of Fungizone and secondary injection of FK23 for the maximal effect was 3 h. Similar augmentation of TNF production was also observed in amphotericin B-primed and FK23-injected mice. Correspondingly, anti-tumour activity of the combined, intravenous injection of Fungizone and FK23 with a 3-h interval was examined. Growth of Meth A fibrosarcoma was clearly inhibited by this combination but not by administration of either one alone. These results suggest that amphotericin B is able to elicit anti-tumour activity, perhaps through activation of the immune system, and in particular augmentation of the induction of endogenous TNF.


LAM S5 is a polysulphated derivative of the glucan laminarian that inhibits basic fibroblast growth factor (bFGF) binding and the bFGF-stimulated proliferation of fetal bovine heart endothelial (FBHE) cells. This report demonstrates that LAM S5 has anti-angiogenic activity, as shown by inhibition of tubule formation by endothelial cells cultured on Matrigel and inhibition of vascularisation of the chick chorioallantoic membrane. In addition, LAM S5 caused a tumour growth delay of the murine RIF-1 tumour of 2.6 days (P = 0.01).