Human ovarian carcinoma cells stably expressing the firefly luciferase gene (SKOV3Luc) were injected i.p. into SCID mice. Tumour growth was evaluated during sustained or intermittent courses of i.p. treatment with paclitaxel (PTX). In vitro bioluminescence strongly correlated with cell survival and cytotoxicity. Bioluminescent imaging detected tumours before their macroscopic appearance and strongly correlated with tumour weight and survival. As compared with intermittent therapy with Taxol®, sustained PTXePC therapy resulted in significant reduction of tumour proliferation, weight and BLI signal intensity, enhanced apoptosis and increased survival times. Our results demonstrate that BLI is a useful tool in the pre-clinical evaluation of therapeutic interventions for ovarian cancer. Moreover, these results provide evidence of enhanced therapeutic efficacy with the sustained PTXePC implant system, which could potentially translate into successful clinical outcomes.We evaluated the pre-clinical efficacy of a novel intraperitoneal (i.p.) sustained-release paclitaxel formulation (PTX Ovarian cancer accounts for the majority of deaths from gynaecological malignancies and remains the fifth most common cancer among women . OvarianePC). Our initial studies demonstrated that PTXePC provided local and controlled release of PTX over several weeks and that it was safer and better tolerated than commercially available PTX formulated in Cremophor EL  chemotherapy and the National Cancer Institute recommends surgery followed by combination of intravenous and i.p. chemotherapy  to determine whether there are therapeutic advantages in providing sustained Establishment of a reliable ovarian cancer xenograft model is an important step in the pre-clinical evaluation of potential treatments. However, monitoring the development and progression of peritoneal disease is difficult. Traditional assessments of efficacy are based on measuring total tumour mass, including areas of necrosis and oedema and do not necessarily evaluate the effects of treatment on the number of viable tumour cells without additional processing and evaluation. In this context, animals must be killed at pre-determined end points, increasing the number of animals required and limiting the possibility of ongoing observation of tumour progression within the same animal. Recently BLI has emerged as a real-time non-invasive method to follow tumour progression  with the Fugene-6 transfection kit . Following transfection, cells were cultured in G418 sulfate (800 μg ml−1) containing medium for 10 days. Surviving colonies were exposed to 1 mM D-luciferin potassium salt  and imaged by IVIS™ Imaging System  for selection of stably co-transfected luciferase/neo clones (SKOV3Luc). Following isolation, SKOV3Luc cells were maintained and propagated in 400 μg ml−1 G418 sulfate-containing medium. SKOV3Luc cells were imaged each week to ensure sustained luciferase expression and studies were commenced 2 months post transfection. Furthermore, to evaluate the stable expression of luciferase over time, SKOV3Luc cells were continuously cultured in G418 sulfate-free media for 3 months and were periodically imaged. Luciferase expression remained stable relative to the initial imaging results.The SKOV3 human ovarian adenocarcinoma cell line was obtained from the American Type Culture Collection . Cells were grown in monolayer cultures in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin  in a humidified atmosphere of 5% COLuc cells were exposed to various PTX concentrations ranging from 0 to 17 080 nM for 24, 48 and 72 h. Cells were then exposed to D-luciferin potassium salt, 1 mM in phosphate-buffered saline  and imaged to determine bioluminescence as described below. Cell viability was determined by the MTT  assay as described earlier (λ=560 nm)( treated ÷ absorbance λ=560 nm) untreated × 100). Linear regression analysis was performed to determine whether a good correlation exists between bioluminescent signal  × 100) and cell survival in treated SKOV3Luc cells. Experiments were performed in triplicate.Exponentially growing SKOV3in vitro imaging, the bioluminescent signal was obtained over a 60-s integration period. For in vivo imaging, mice were pre-anaesthetised  and were administered D-luciferin potassium salt in saline (100 mg kg−1) intraperitoneally. Animals were then placed in an air-tight box in the imaging chamber  at a distance allowing for whole body imaging in a single field of view. Gray-scale images were initially obtained to provide an anatomical reference for the bioluminescent images, which were then acquired over a 5-min integration time. Regions of interest covering the entire peritoneal cavity were selected, including tumours and total photon counts were determined. Bioluminescent images were collected at various time points throughout the duration of the study.Bioluminescent imaging was performed with the IVIS™ Imaging System, composed of a high sensitivity cooled charge-coupled camera mounted in a light-tight box . The commercially available Living Image™ software  was used to collect and analyse images. For 7 SKOV3Luc cells, suspended in 200 μl of RPMI-1640 medium. Animals were housed under sterile conditions in microisolator cages, fed standard chow diet with water ad libitum and maintained on an automatic 12-h light cycle at 22–24°C. All studies were conducted using sterile techniques and in accordance with the guidelines of the University Health Network Animal Care Committee and the Canadian Animal Care Council.Female SCID mice , obtained from the University Health Network animal colony  were injected intraperitoneally with 1 × 10−1 total dose over 3 weeks) either as intermittent therapy (i.p. bolus injections of 20 mg kg−1 Taxol® on a q7d × 3 schedule) or sustained therapy  and were compared with controls (drug-free ePC implant). Prior to treatment initiation, release from PTXePC was confirmed both in vitro and in vivo as described earlier , were separated into the various groups as described above (n=12/each group). Mice were imaged every 2 days post SKOV3Luc tumour inoculation, at treatment initiation, 24-h post-treatment initiation and every 2–3 days throughout the course of treatment until the termination of the study. Each week , three mice from each group were randomly selected and killed for analysis of tumours and tissues. Owing to rapid disease progression, a maximum 4-week monitoring period was established. Animals were monitored daily for normal activity, abdominal swelling (peritonitis), weight loss, muscle wasting, abdominal distension, and under-conditioning to assess treatment-related toxicities and disease progression. To monitor abdominal distension, we utilised an autoclavable, fabric measuring tape to measure abdominal girth two times weekly, commencing at the day of tumour inoculation. To assess under-conditioning and muscle wasting, we used the ‘body conditioning scoring’ system as described earlier  in 0.01 N HCl (pH 2.0) for 15 min at 37°C. Endogenous peroxidase and biotin activity were blocked using 3% hydrogen peroxide and avidin/biotin blocking kit . For the assessment of proliferation, sections were incubated at room temperature with the Ki-67 monoclonal antibody , 1/50 dilution for 1 h. For the assessment of apoptosis, sections were incubated with active caspase 3 (casp3) antibody , 1/50 dilution overnight, or with biotin-nucleotide cocktail and DNA polymerase 1  for 1 h at 37°C for in situ terminal deoxytransferase-mediated dUTP nick-end labeling (TUNEL). Colour development was done with freshly prepared NovaRed solution . Sections were counterstained lightly with Mayer's haematoxylin, dehydrated in alcohols, cleared in xylene and mounted in Permount . Nuclei that stained brownish-red were scored as positive and those that stained blue were scored as negative.Paraffin sections were dewaxed in xylene, passaged through graded alcohols and rinsed in distilled water. Sections were immersed in 10 mTumour sections were imaged using a Nikon Coolpix 990 colour camera mounted on a Nikon Eclipse e400 microscope (Japan). At least 10 fields ( × 400) were randomly selected from each slide. Positively and negatively stained nuclear areas were collected by using Image J analysis software . Ki-67, Casp3 and TUNEL-labeling indices were determined as the ratio of areas occupied by the positively stained tumour cell nuclei relative to all tumour cell nuclei.t-test of unequal variance for comparison between groups. Regression analysis was performed to establish correlations and the log-rank test for survival analysis. Differences were considered statistically significant at P<0.05.Data are expressed as means±s.e. Data were analysed using one-way ANOVA and the paired Student's Luc cell survival in a concentration and time-dependent manner as evaluated by BLI and MTT. As duration of exposure was increased, cell survival decreased and the IC50 values were 683±72, 137±29 and 27±12 nM PTX at 24, 48 and 72 h, respectively. The bioluminescent signal (% control) significantly correlated with the MTT assessment of cell viability .PTX inhibited SKOV3iability . Furthervs intermittent intraperitoneal PTX administration was examined in vivo in the SKOV3Luc xenograft model of human ovarian cancer in SCID mice. Sustained intraperitoneal PTX delivery with PTXePC resulted in significant reduction of tumour growth, whereas intermittent therapy with Taxol had no significant effect on tumour reduction as measured by bioluminescent imaging and tumour weight  and bioluminescence signal .The effect of sustained r weight . Biolumir weight . The dif animals  and 4. PePC treatment significantly increased the proportion of apoptotic tumour cells, whereas intermittent Taxol therapy had no effect . However, much greater concentrations were accomplished in the peritoneal cavity at the tumour site. Therefore, PTXePC therapy achieved augmented tumour targeting and diminished systemic toxicity. Alternatively, peak plasma and liver concentration levels have been reported to be about 17 μg ml−1 and 60 μg g−1 respectively, following a single i.p. bolus doses of PTX (20 mg kg−1); concentrations associated with numerous toxicities (ePC was also reflected by the lack of significant changes in body weight. Indeed, we have previously demonstrated favourable toxicity profiles with PTXePC and augmented toxicity with Taxol (In addition to diminished tumour repopulation, this enhanced tumour responsiveness with PTXur cells . Indeed,Localised chemotherapy alleviates systemic toxicities and has demonstrated survival advantages in advanced ovarian cancer patients; however, catheter-related complications currently remain a limitation in the clinical application of intraperitoneal chemotherapy. Our drug delivery system may be a solution to the problems encountered with current intraperitoneal chemotherapy administration practices and allow further exploration of localised drug delivery. Moreover, our formulation provides sustained-release of PTX, which may further improve efficacy by diminishing the rates of tumour repopulation and by enhancing tumour cell apoptosis. We also demonstrate that bioluminescent imaging enables earlier detection of tumours than traditional methods and that signal intensity correlates with tumour growth. Therefore, bioluminescent imaging is a non-invasive and rapid approach for the monitoring of experimental tumours and should be further utilised in the pre-clinical evaluation of therapeutic interventions for ovarian cancer.


Better understanding of the pathways regulating proliferation and metastasis of cancer cells has led to the development of novel molecular-targeted therapies. The number of molecular-targeted agents approved for use in the clinic is growing, with many more in clinical trials. Most of these compounds can be broadly classified into two main categories: monoclonal antibodies and small-molecule tyrosine kinase inhibitors. The pathological processes targeted include vascular endothelial growth factor-dependent tumour angiogenesis and epidermal growth factor receptor-dependent tumour cell proliferation and survival. Unlike conventional chemotherapy, molecular-targeted agents offer the potential advantages of a relatively high therapeutic window and use in combination with other anticancer strategies without overlapping toxicity. It is hoped that these drugs will become valuable therapeutic tools within the multimodal approach to treating cancer. Recent progress in targeted antitumour therapy is discussed, with a focus on antiangiogenesis. Front-line therapy for cancer not accessible to surgical intervention has for the last 30 years remained a combination of chemo- and radiotherapy. Despite recent advances in patient survival resulting from the development of newer generation cytotoxic agents such as the taxanes and gemcitabine, the overall prognosis for patients with most common cancers remains poor. Failure to improve conventional cytotoxic therapy over a period of several decades has led cancer researchers to search for completely novel approaches to treat cancer.The present time is an exciting one for those involved in cancer research and treatment, as several of the strategies embarked upon a decade ago to enable targeting of tumour therapy are now achieving fruition . Such apAs noted in the introduction, the mainstay of cancer chemotherapy in recent decades has been the use of cytotoxic agents. In effect, one attempts to identify an agent that kills tumour in preference to normal cells. Despite expenditure of vast effort on the development of cytotoxic agents, progress in recent years has been slow. In particular, the narrow therapeutic index of cytotoxic drugs and the emergence of drug resistance mean an unfavourable prognosis for many cancer patients.The majority of human cancers arise from epithelial tissues in which the epithelial cells are undergoing continuous replication to maintain the protective layer of cells in contact with the environment. Examples include the lining of the lung and digestive tract and in the epithelium of secretory organs such as the breast and prostate. The malignant transformation of normal human epithelial cells into carcinoma cancer cells is a complex, multistep process. An essential change required for the development of all cancers is mutation in appropriate genes. There are usually several genetic changes involving, for example, proto-oncogenes, tumour-suppressor genes and genes that regulate cell cycle events. Once a cell has lost normal control of its growth and division, it expands and multiplies to form the initial primary tumour. At this stage, the tumour is fed by diffusion from nearby blood vessels; however, as the tumour cells increase in number they eventually become starved of nutrients such as oxygen and glucose. At this point during the early development of a tumour, the angiogenic switch occurs, and this is of particular relevance to therapeutic approaches that target angiogenesis . The angin vitro, reliable and reproducible in vivo angiogenesis assays were developed and at the molecular level, potent peptide angiogenic factors such as fibroblast growth factor and VEGF were identified. Two key tenets supported early research into tumour antiangiogenesis. Firstly, as angiogenesis is largely absent in the healthy adult, it was widely held that an antiangiogenic approach would be without side effects, and secondly, the fact that the endothelial cells involved in angiogenesis are normal cells meant that they did not suffer the genome instability seen in carcinoma cells and as such were unlikely to develop drug resistance. Drug resistance had long proven a problem in targeting carcinoma cells directly.That tumours require new vasculature to grow and that inhibition of the growth of tumour vasculature or antiangiogenesis could be an effective anticancer strategy has been the subject of intense research. The idea had been around for some time, but was popularised in the 1990s by researchers such as Judah Folkman, who collected the evidence arguing that angiogenesis is needed for tumour growth in a cogent article entitled ‘What is the evidence that growth of solid tumours is angiogenesis dependent?’ . An incrMost deaths from cancer result from metastases and metastatic lesions are an important target of antiangiogenic therapy. The problem with metastases is the difficulty of treatment. Surgery is rarely an option once the tumour has spread beyond a limited area and the same applies to radiotherapy, hence the attraction of systemic therapy and the sustained efforts to develop it.in vivo. The fact that VEGF was (until recently) thought to be a growth factor specific for endothelium and not widely expressed outside of tissue undergoing active angiogenesis  identified VEGF as a key molecular target for antiangiogenic therapy. It is now generally accepted that VEGF-induced angiogenesis and hyperpermeability are mediated via binding to the VEGF receptor-2 (VEGFR-2) located on the surface of vascular endothelial cells. Binding causes VEGFR-2 dimerisation and induces tyrosine kinase (TK)-mediated autophosphorylation, which in turn leads to activation of the relevant downstream signalling pathways.A large body of evidence has confirmed VEGF as a key regulator of tumour angiogenesis  and the related receptor HER-2, Bcr-Abl, c-Kit, platelet-derived growth factor receptor (PDGFR) and CD20. Most of the molecular-targeted agents currently approved or undergoing clinical evaluation can be broadly classified into two main categories: monoclonal antibodies or small molecule TK inhibitors . MonocloMolecular-targeted agents offer a spectrum of selectivity, from single-target monoclonal antibodies such as bevacizumab  without significant overlapping toxicity.Ongoing use of approved agents, as well as further evaluation of new drugs, will reveal to what extent the clinical promise of targeted therapy is to be fulfilled. Most recently, the success with bevacizumab in advanced metastatic colorectal cancer was an encouraging demonstration of proof-of-principle of an anti-VEGF approach. Administration of bevacizumab with irinotecan, fluorouracil and leucovorin (IFL) gave a median survival of 20.3 months against 15.6 months for patients receiving IFL alone . ClearlyN-acetylcysteine (‘Parvolex’) (Impressive as the clinical results with bevacizumab are it is starkly apparent that at best it inhibits but does not halt tumour growth, so there remains much to do. Why is the therapy not affecting more long-term survivors or even cures? The reasons are no doubt complex. One possibility is that VEGF is not the sole angiogenic factor produced by the tumour. Indeed, many publications have shown that most tumours express multiple angiogenic factors . DiffereA complementary anticancer strategy that also targets the endothelium is tumour vascular targeting . DestrucThe ultimate success of any targeted agent will be measured by whether or not it provides clinical benefit to patients. In this respect, the current design of clinical trials involving antiangiogenic agents may be improved by the development of rapid and effective biomarkers to establish drug dosage and monitor clinical response. The clinical utility of targeted agents is now established for both tumour and tumour blood vessel targets, with a number of agents already approved  and othe


In order to determine the mechanisms of relapse following response to endocrine therapy, we have measured the oestrogen receptor (RE) content of biopsies of breast cancer in patients receiving various types of endocrine treatment. RE content fell in responding (means of 260.2 to 12 fmol/mg protein) and in nonresponding (means of 155.1 to 31.8 fmol/mg protein) patients who had measurable receptor at the start of treatment. Some of these patients, and a further group of responders to endocrine therapy, were monitored until relapse. Tumour biopsies at the time of relapse showed that 10/14 tumour samples contained significant RE  after successful endocrine therapy. No relationship could be found between RE content and plasma gonadotrophin or steroid-hormone concentration, but the fall in RE content correlated with reduced numbers of tumour cells in the biopsy. These results indicate that relapse following successful endocrine therapy in breast cancer does not appear to be due to the emergence of RE-negative tumour cells. The fall in RE content during response to endocrine therapy may be due to reduced tumour-cell content of the biopsy.


This effect we have shown to be solely in the 19s fraction after Sephadex G-200 separation.Cell-free lymph from immunized rats was shown to be cytotoxic to the tumour cells This 19s fraction conferred partial protection to tumour bearing mice when given shortly after transplantation, but had negligible effect against late well established tumour.


Mice were implanted with a lymphosarcoma on either the thorax or leg. Some of these tumours were given a single dose of 240 kV x-rays on reaching a predetermined size, whilst others were left unirradiated.Metastases developed in specific lymph nodes, depending on the position of the transplanted tumour, and were larger if the tumour had been irradiated than if not. The growth rate of metastases in the ipsilateral axillary lymph node was faster than that of the transplanted tumours, irrespective of the radiation dose the tumour had received.


Delay between initiation and promotion on mouse skin was in 1949 reported by Berenblum and Shubik not to affect tumour yields, and this led to the important concept of the irreversibility of initiation and stimulated the development of multistage models. Subsequent reports have, however, suggested that delay does decrease tumour yields, and this is confirmed by the present study of 2200 mice initiated at 8, 48, or 68 weeks with 10, 30, 100, or 300 microgram of DMBA and promoted by a standard dose of TPA for 15 weeks, after various delays. However, our data suggest that the decrease in tumour yields is chiefly or wholly due to a reduction, among ageing mice, of the ability to respond to promoters, and not to any substantial loss of initiated cells, for late initiation with immediate promotion also yielded a less rapid response than early initiation with immediate promotion. Interpretation of all such studies is complicated by the few weeks that the skin needs to repair ulceration and other damage induced by the higher doses of DMBA, for if promotion with TPA begins before such repair is complete the tumour yield may be misleadingly increased.


RENCA cells were implanted into the mouse cremaster muscle and the trafficking of syngeneic lymphocyte subpopulations, derived from naive and ‘immune’ animals, into both the RENCA tumour and the surrounding normal cremaster muscle microcirculation was measured by in vivo microscopy. Fluorescently labelled CD4+ and CD8+ T lymphocytes taken from the spleens of naive mice or mice previously immunized with RENCA-IL-2 were injected systemically into tumour-bearer mice. Naive effector cells migrated to, and flowed through both the tumour and the normal microcirculation, with negligible adhesion. However we observed the selective recruitment, localization and arrest of immune CD4+ and CD8+ T lymphocytes (P < 0.05) into the tumour microcirculation, and in some instances the subsequent extravasation of cells into the tumour interstitium. Lymphocyte rolling by ‘immune’ CD4+ and CD8+ T-cells in the tumour microcirculation was greatly reduced, suggesting impaired adhesion molecule expression on the tumour endothelium. This study clearly demonstrates, by direct in vivo microscopy assessment, the localization of effector cells, CD4+ and CD8+ lymphocytes into tumours. © 2000 Cancer Research CampaignRENCA-IL-2  cells were rejected by immunocompetent (but not T-cell deficient) Balb/c mice, which developed ‘immunity’ to subsequent parental RENCA tumour cell challenge. Splenocytes adoptively transferred this immunity. CD4


Single-cell suspensions prepared by enzymatic disaggregation of an immunogenic 3-methylcholanthrene-induced sarcoma (Mc40A) contain a significant proportion of infiltrating leucocytes (approximately 42%), comprising T lymphocytes, macrophages and non-phagocytic FcR+ lymphoid-like cells. Tumour-infiltrating lymphocytes (TIL) were isolated and purified by successive passage over Sephadex G-10 columns and their cytotoxic activity in vitro compared with that of lymphoid cells from normal rats and from tumour-bearers at different times after implantation. For this purpose, surviving target cells were quantified by incorporation of the gamma-emitting analogue of methionine, 75Sel-methionine, in a 48-h assay which detected both cytotoxic and cytostatic effects. The reactivity of TIL, which was consistently demonstrable from 11 days after tumour transplantation, was essentially similar to that of normal splenic lymphocytes in magnitude and specificity. Reciprocal cytotoxicity tests using TIL and cultured targets from an antigenically unrelated tumour of similar aetiology (Mc57) showed that the manifestation of TIL cytotoxicity was determined, not by the tumour of origin, but by the susceptibility of the target cells. Evidence that the effector function of TIL was mediated in part by natural killer (NK) cells was derived from concurrent experiments using human myeloid cells (K562) as targets in an 18h 51Cr-release assay. In this system the level of NK activity was critically dependent on the numbers of tumour cells in the TIL population; contamination in excess of 2% gave rise to dose-dependent inhibition of NK function. The results show that within a progressively growing tumour known to possess rejection antigens, NK reactivity was detected in the absence of a demonstrable tumour-specific cytotoxic component.


Metastatic liver involvement was observed initially within a week post induction, as areas approximately 100 μm in diameter not perfused by the casting resin. On histology these spaces corresponded to tumour cell aggregates. The following weeks highlighted the angiogenesis phase of these tumours as they received a vascular supply from adjacent hepatic sinusoids. Direct sinusoidal supply of metastases was maintained throughout tumour growth. At the tumour periphery most sinusoids were compressed to form a sheath demarcating the tumour from the hepatic vasculature. No direct supply from the hepatic artery or the portal vein was observed. Dilated vessels termed vascular lakes dominated the complex microvascular architecture of the tumours, most tapering as they traversed towards the periphery. Four vascular branching patterns could be identified as true loops, bifurcations and trifurcations, spirals and capillary networks. The most significant observation in this study was the direct sinusoidal supply of metastases, together with the vascular lakes and the peripheral sinusoidal sheaths of the tumour microculature.Development of effective treatment for hepatic metastases can be initiated by a better understanding of tumour vasculature and blood supply. This study was designed to characterise the microvascular architecture of hepatic metastases and observe the source of contributory blood supply from the host. Metastases were induced in mice by an intrasplenic injection of colon carcinoma cells (10


Splenocytes from inbred Wistar rats bearing a syngeneic squamous cell carcinoma (Spl) were fractionated by several techniques to characterize the lymphoid cells cytotoxic to the tumour in vitro. The anti-tumour cytotoxicity is presumably mediated primarily by T lymphocytes because it was greatly reduced by removal of T lymphocytes with heterologous anti-T serum plus complement but not by removal of other cell types. Cytotoxicity could be blocked at the tumour cell but not at the effector cell by sera taken late in tumour growth. Sera taken earlier in tumour growth could induce cytolysis of tumour cells by normal splenocytes but only if the tumour cells were treated with serum and washed before addition of the effector cells. Although splenocytes from normal and tumour-bearing rats were equally effective at lysing antibody-coated target cells it is unlikely that this mechanism is important in vivo as sera from early in tumour growth onwards contained factors (immune complexes?) which inhibited antibody-induced lymphocytolysis.


In tumour regrowth-delay experiments, the analysis of results in the higher dose ranges may be complicated by a dose-dependent proportion of non-recurrent (cured) tumours, whose inclusion in the analysis is not straightforward. A study of the relation of growth delay to cure has been carried out using a model of tumour response which assumes Poisson (single-cell) cure statistics and exponential regrowth kinetics of recurrent tumours, and which makes use of Monte Carlo simulation techniques to represent the effects of inter-tumour heterogeneity. This approach yields correction factors compensating for tumour cures in growth-delay experiments. For homogeneous tumour systems  these corrections are small and not significantly different from corrections obtained using the "long delay" procedure suggested previously . For heterogeneous systems, however, correction factors increase with the heterogeneity of the system, and may become quite large. It is concluded that the quantitative assessment of heterogeneity is required, and a possible approach to this is suggested. Should evaluation of heterogeneity prove feasible, it will allow more efficient use of tumour-response data, and may permit realistic estimates of clonogenic cell survival in situ.


An analysis of the constituents of the plasma membrane of a methylcholanthrene-induced sarcoma (the MC1 tumour) in a hooded rat revealed four tumour-associated macromolecules. Two of these were antigenic in the syngeneic host, one was unique to the MC1 tumour and could not be detected in embryo tissue and has the properties to be expected from the well established tumour-specific transplantation-type antigen while the other, referred to as OEA I, was present in all rat sarcomata tested as well as in early embryos. Two other embryonic components were detected in the sarcoma but these were not immunogenic in the rat. The properties of these tumour-associated “antigens” in the membrane of rat sarcomata are summarized below: [Table: see text]


Enzymatic activity of SOD was also increased in oxykine group. Arising tumour cells in gliadin and saline groups acquired metastatic phenotype, but those in oxykine group showed reduced metastatic ability. These results suggested that the orally active SOD derivative prevented tumour progression promoted by inflammation, which is thought to be through scavenging inflammatory cell-derived superoxide anion.Weakly tumorigenic and nonmetastatic QR-32 cells derived from a fibrosarcoma in C57BL6 mouse are converted to malignant cells once they have grown after being coimplanted with a gelatine sponge which induces inflammation. We administered a newly developed peroral superoxide dismutase (SOD), oxykine, and as control vehicle, gliadin and saline, starting 2 days before the coimplantation and continued daily throughout the experiment. In the oxykine group, tumour incidence was lower (41%) than in the gliadin or saline group . The inhibitory effect of oxykine was lost when an individual component of oxykine was administered, that is, SOD alone and gliadin alone. The effect was also abolished when administered by intraperitoneal route. When perfused  Evidence has been accumulated that many of the cell alterations seen in normal ageing and in various diseases including cancer are due to oxidative damage by active oxygen species . Oxygen 2−) to produce hydrogen peroxide and O2. Superoxide anion is one of the reduced oxygen species generated in cellular metabolism or produced by inflammatory cells at their respiratory burst.Most aerobic cells have an enzymatic system to eliminate active oxygen species, because some of these active species are toxic to host. Superoxide dismutases (SODs), catalase, and glutathione peroxidase comprise the major defence system against oxygen toxicity . Superoxin vivo  for comparing SOD amounts before and after transformation  which not only prevents gastric digestion  whereas they developed tumours after coimplantation with a foreign body, gelatine sponge  and progression of tumour cells (acquisition of metastatic phenotype). We herein reported that oxykine reduced primary tumour growth and prevented the acquisition of metastatic property of tumour cells through suppression of superoxide anion at the tumour-growing sites.−1. It was diluted with PBS just before usage and kept on ice until administration. The dose was fixed at 10 mg kg−1, and administered intragastrically.Oxykine®, melon-derived SOD and gliadin were provided by Asama Chemical Inc., Tokyo, Japan. The SOD activity of oxykine was 1000 U gin vitro to quercetin, which gave rise to a number of random subclones  were obtained from Nippon SLC  and used for the experiments. All the mice were maintained in the complete barrier condition, lit from 0700 to 1900, at 23 × 3°C and 50 × 10% humidity, fed with mouse diet  and UV-irradiated water in the germ-free section of Institute for Animal Experimentation, Hokkaido University Graduate School of Medicine. Diet and tap water were available The experimental protocol was approved by the Committee of Institute for Animal Experimentation, Hokkaido University Graduate School of Medicine (#01139).3 piece; Spongel, Yamanouchi Pharm., Japan) was inserted and the wound was closed with clips. Then QR-32 tumour cells (1 × 105 cells/0.1 ml) were immediately injected into the inserted gelatine sponge  into normal C57BL/6 mice. On day 25, the mice were killed and metastatic nodules at the surface of the lungs or other organs were counted macroscopically.The mice were divided randomly into three groups and treated with oral administration of oxykine or gliadin at a dose of 10 mg kgThe gelatin sponge pieces subcutaneously injected into the mice treated with oxykine, gliadin, melon-SOD or saline were removed and digested with 0.2% collagenase in serum-free MEM medium for a few minutes at 37°C. After collecting all the infiltrated cells by centrifugation, we counted total number of the cells per piece of gelatin sponge. We also counted differential counts of more than 200 cells in smear preparations of the collected cells stained with May-Gruenwald's and Giemsa solution . Mean percentages of differential cells were obtained from the mean values of independent counts by two pathologists.−1) in Hank's balanced salt solution . All unreacted NBT was removed from the tumour masses by washing with HBSS. The NBT-perfused tumour mass was photographed and then fixed with 10% (wt vol−1) zinc/formalin for histologic examination of formazan deposits. The procedure followed the method described in a previous paper with slight modifications  activity was determined by using a β-butyl hydroperoxide as substrate. One unit of the enzyme activity was defined as 1 μmol NADPH oxidised min−1 at 36°C. The enzyme activities of Mn-SOD, CuZn-SOD, catalase and GPx were expressed as U mg−1 protein. Protein concentration was estimated by the Lowry method  or intravenous (1 × 106 cells) injection into normal syngeneic C57BL/6 mice  did not develop tumours or form metastasis after subcutaneous  statuses in both tumour cells and the inflammatory cells both of which had infiltrated into gelatin sponge. After harvesting those cells by collagenase digestion, we compared cytosolic ROS levels by using an ROS probe, 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydro-fluorescein diacetate (CM-H2DCFDA) and measured intracellular ROS levels by flow cytometry. Mean fluorescence intensities of the cells harvested from the gelatin sponge in the mice orally treated with oxykine, melon-SOD (main component of oxykine), gliadin or saline were 37.4 × 8.6, 42.5 × 13.6, 49.9 × 22.2, 47.5 × 14.2, respectively. We detected positive tendency to decrease the production of ROS in the oxykine-treated group as compared to those in other groups; however, the difference was not significant. We then compared the antioxidative enzyme activities such as those of SOD, glutathione peroxidase and catalase in the serum or tumour tissues after oral administration of oxykine, gliadin or melon-SOD . We founWhen we administered oxykine, melon-SOD, gliadin or saline to mice via intraperitoneal route, the effect of oxykine as observed in peroral administration was lost . This fiin vitro culture cell lines from the arising tumours in the mice treated with oxykine, gliadin or saline, and designated them as QRsP/OK, QRsP/GD and QRsP tumour lines, respectively. As P<0.001). At the time of killing, there was no evidence of typical spontaneous metastasis.It is the advantage of this model that we can determine whether the arising tumour cells acquire metastatic ability without inflammatory cells, because the tumour cell lines had been established by culturing the cells from tumours arisen in individual mice and were originally their metastatic potential is examined in another normal syngeneic mice (P<0.001). Although there was no reduction of lung metastasis with control vehicle or gliadin treatment, it was suppressed by administration of oxykine (97% inhibition).Subcutaneously injected gelatin sponge pieces into the mice with administration of oxykine, gliadin, melon-SOD or saline were removed and the exact number of infiltrated cells was counted per gelatin sponge. −1 day−1 for 27 days) did not cause either any obvious side effect such as weight loss, or alteration in the appearance or behaviour of the tumour-bearing mice during the observation period. The data of average body weight are shown in The application of oxykine or gliadin -derived SOD. The C. melo LC. derived SOD has an SOD activity which is more than five times that of classical melon species, charentais  intravenous or subcutaneous administration of recombinant human SOD which substitutes specific amino acid for stable one , and mosarentais .in situ superoxide production levels in each treatment in Niitsu and his colleagues have discovered an inhibitory effect of SOD on both experimental and spontaneous pulmonary metastasis in murine models (2−, which in tern downmodulates signal transduction and/or activation of transcription factors to suppress cell growth (in vivo. Indeed, the QR-32 tumour cells are highly sensitive to LAK cells (de novo SOD at the tumour-growing sites. In our experiments, we revealed that Mn-SOD was induced in tumour cells only by oxykine formulation (We also observed inhibition of the primary tumour growth in the group with oxykine administration. There are three possible explanations for this. One is that the elevated levels of SOD might lower intracellular levels of Ol growth . Irani emulation . Howevermulation . TherefoThe formation of human cancer involves a multistage process, that is, initiation, promotion and tumour progression. Oxidative stress is considered to contribute to the whole process from carcinogenesis through induction of mutations or upregulation of cell growth in target cells . In this


In vivo interactions are therefore of critical importance in determining this high potency. The pharmacokinetics of m-THPC in a rat tumour model was determined using 14C m-THPC in an LSBD1 fibrosarcoma implanted into BDIX rats. The photodynamic therapy (PDT) efficacy was determined at different drug administrations to light intervals and correlated with the tumour and plasma pharmacokinetic data. The plasma pharmacokinetics of m-THPC can be interpreted by compartmental analysis as having three half-lives of 0.46, 6.91 and 82.5 h, with a small initial volume of distribution, suggesting retention in the vascular compartment. Tissues of the reticuloendothelial system showed high accumulation of m-THPC, particularly the liver. PDT efficacy of m-THPC over the same time course seemed to exhibit two peaks of activity (2 and 24 h), in terms of tumour growth delay with the peak at 24 h postinjection correlating to the maximum tumour concentration. Investigation on tumour cells isolated from m-THPC-treated tumours suggested that the peak PDT activity at 2 h represents an effect on the vasculature while the peak at 24 h shows a more direct response. These results indicate that the in vivo PDT effect of m-THPC occurs via several mechanisms. It is based on a tumour accumulation of a photosensitiser, which, when activated by light, results in tumour destruction via reactive oxygen species . The selm-Tetra (hydroxyphenyl)chlorin (m-THPC), a second-generation photosensitiser, has already been shown to be more potent than Photofrin (PII). It has been suggested that it is up to 200 times more powerful (−1 compared to 10 mg kg−1 for PII) and the lower light doses necessary (30 J cm−1 rather than 150 J cm−1) to produce similar PDT results. However, the reason for this effectiveness remains unresolved even considering the advantageous photoproperties of increased molar absorption coefficient and a favourable shift in the wavelength maximum (652 nm rather than 630 nm) chlorin and 14C m-THPC were supplied by Scotia QuantaNova. The sensitiser was dissolved as recommended for intravenous injection in a mixture of polyethylene glycol 400 : ethanol : water (3 : 2 : 5 by volume). Radioactive sensitiser was diluted with unlabelled m-THPC to a specific activity of 16.9 mCi mmol−1 and a final concentration of 0.6 mg ml−1. Unlabelled m-THPC was also prepared as a 0.6 mg ml−1 solution and the solutions were stored in 0.5 ml aliquots at −20°C.Animal experiments were performed under protocols approved by the local experimental animal welfare committee and conformed to National and European regulations for animal experimentation. Experiments were covered by Home Office premises, project and personal licenses as well as keeping within the UKCCCR guidelines on use of animals for experimental neoplasia (1), which grows as a sphere causing no skin ulceration and no attachment to the muscle below. The tumour was implanted as a subcutaneous fragment (1 mm) when the rats weighed 300–400 g chlorin (0.3 mg kg−1) was injected intravenously via the penile vein.Rats were weighed and tumours measured daily. Measurements were taken on three perpendicular axes using callipers to calculate the mean tumour diameter. When tumours had reached a mean diameter of 8–10 mm, approximately 10 days after implantation, they were suitable for PDT treatment and were entered into the study. 14C m-THPC or unlabelled m-THPC was administered at 0, 2, 4, 6, 18, 24, 48, 72 or 96 h before the tumour was predicted to reach 8–10 mm in diameter. This was determined from the known tumour growth kinetics. In addition, a 168-h time point was used in the pharmacokinetic study that had a slightly different protocol, consisting of m-THPC administration when the tumour was 8–10 mm. All procedures were carried out using UKCCCR guidelines (−1) and centrifuged to separate the plasma. Three animals were used per time point and all samples were stored at −20°C. Procedures were carried out using UKCCCR guidelines  and skin . Dissection was carried out under subdued light, and all tissues were washed in isotonic saline and blotted dry. Triplicate samples of each tissue (90–110 mg) were weighed directly into scintillation vials. Blood samples (2.5 ml) were collected by heart puncture . The samples were then diluted with an equal volume of heparin solution   were added to each vial followed by vigorous shaking. Radioactivity was determined using a Packard Liquid Scintillation Analyzer (1900TR). An internal radiolabelled standard, 14C n-hexadecane was used to determine the counting efficiency. The plasma samples were analysed using 200 μl aliquots  mixed with 10 ml scintillation fluid and counted as above.Each tissue sample was digested by addition of 1 ml of 1 For each animal, the mean tissue concentration was calculated. The mean and the standard deviation (s.d.) for all three animals at each time point were calculated. The plasma pharmacokinetics was analysed by compartmental and noncompartmental mathematical methods .μm optical fibre with a 0.5 cm diffusing tip  and the animals were killed at 2 and 24 h together with a control animal. Procedures were carried out using UKCCCR guidelines . The collagenase II mixture was poured off and the remaining cells resuspended in 10 ml of phosphate-buffered saline (PBS). The cell suspension was then passed through a 100 μm nylon filter mesh and centrifuged as above. The cells were again resuspended in 10 ml of PBS and centrifuged. This was repeated until the suspension was clear, then the cells were suspended in 5 ml Dubecco's modified Eagle's medium (DMEM) containing 10% FCS and 1% glutamine. The concentration of cells in the suspension was determined using a haemocytometer. Cells were seeded at 1 × 105 per well of a 96-well plate, in 200 μl of medium, and left for 4 h to allow cell attachment. Immediately before illumination, the medium was removed from the cells and replaced with PBS.The tumour cell suspension was passed through a 150 −2, by varying the illumination time (0–16 min) and then incubated for 18–24 h at 37°C before determining cell survival by the MTT assay (The plates were irradiated using a xenon arc lamp fitted with a 652 nm filter (±15 nm). The cells were treated with light doses of 0, 2.5, 5, 10, 20 and 40 J cmTT assay .Statistical analysis was carried out using analysis of variance in the computer program SAS 6.12 system for Windows™ .−1). The plasma levels were initially high (9.37 μg ml−1) and appear to declined exponentially as would normally be expected for an intravenous injection. The clearance from plasma, however, did not follow a simple monoexponential decay but could be fitted to multiple exponentials, and because of this the data were analysed by both a noncompartmental and a compartmental approach  was estimated by extrapolation of the log concentration vs time graph back to zero.The plasma concentration of m-THPC at time zero (k′∞) was estimated between 72 and 168 h.The slope of the log linear (elimination) phase (vs time graph (AUC) was calculated from time zero to the last time point (T(last)) by the trapezoidal rule and from T(last) to infinity as Area=C(last)/k′, where C(last) is the plasma concentration at the last time point (168 h).The area under the concentration vs time curve  was calculated from time zero to T(last) by the trapezoidal rule and T(last) to infinity as Area=C(last)/(k′)2.The area under the (concentration × time) CL=DOSE/AUC.Plasma clearance Mean residence time MRT=AUMC/AUC.Vd=CL × MRT.Volume of distribution T1/2=MRT × ln(2).Half-life Kel =1/MRT.Elimination rate constant For the noncompartmental approach, the calculation method was as follows.−1 and a biological half-life of 22.6 h.C0), was estimated to be 32.0 ml kg−1. The phases of the three compartments, however, have volumes of distribution of 50.9, 89.3 and 2500 ml kg−1 .From the compartmental analysis, the initial volume of distribution of the central compartment, calculated from the initial plasma concentration . The slow elimination is probably due to slow diffusion out of these tissues. This indicates another possibly longer half-life for m-THPC, which was not encountered in the plasma pharmacokinetics during this time course.The elimination rates from the tissues . However, the 2 h interval was significantly different to all time points except 4 and 24 h (P<0.05). Hence, there were two peaks in tumour growth delay, an early peak around 2–4 h followed by a second peak around 24 h. At the time intervals that caused significant tumour growth delay, there were periods of tumour stasis that lasted up to 5 days after the initial regression; this was then followed by a period of increased growth at a slower rate than the controls (data not shown).Photodynamic therapy efficacy peaked at 2–4 h postadministration with an apparent second peak at 24 h. There was no significant difference between treatments at 0, 6, 48, 72 and 96 h for the diode laser , as m-THPC is known to remain in cells even if serum is present extracellularly .The LSBDin vivo interactions, such as aggregation and protein binding (14C) m-THPC was used in this study to avoid the problems encountered with noninvasive induced fluorescence determination , which is small compared to the blood volume of a rat (50–70 ml kg−1). m-Tetra (hydrophenyl)chlorin may be initially retained in the vascular system, possibly due to protein binding or precipitation  could be due to the concentration of m-THPC being diluted due to the rapid growth of the tumour. The LSBD1 tumour in this model has a doubling time of approximately 1.5–2 days. These kinetic data are similar to those seen in other tumours and animal models. μg g−1 and μg g−1. High levels of m-THPC in the tumour would suggest a good PDT response, although the intracellular localisation of the photosensitiser is critically important. The tumour concentration rises quickly in the first 6 h with its concentration staying above 0.4 μg g−1 and the elimination profile was almost identical to that of the tumour (data not shown). This follows observations that tissues of the reticuloendothelium system accumulate high concentrations of photosensitisers  was that there were two peaks of tumour growth delay (2 and 24 h). In studies in human mesothelioma xenografts, a similar early peak of PDT effect was seen at 3 h mice (μg g−1 at 2 h and 0.54±0.15 μg g−1 at 24 h) produce similar tumour growth delay (The pattern of efficacy does not follow the pattern of m-THPC concentration in the tumour. Significantly different drug concentrations in the tumour (0.29±0.03 th delay . Similarth delay .1 tumour at 2 and 24 h was examined by assessing tumour cell survival after administration of m-THPC in vivo, followed by cell isolation and light exposure in vitro.The mechanisms involved in the initiation of tumour destruction by PDT are difficult to distinguish and are thought to be dependent on the localisation of the photosensitiser due to the small diffusion distance of the reactive oxygen species (in vivo the effects are relatively similar, indicating either more m-THPC present in the cell or a specific localisation within the cell. Localisation studies of m-THPC have shown strong fluorescence in the vasculature walls at 1 h, while at 24 h m-THPC is still present in the vasculature but also localised intracellularly (The cells extracted 24 h after injection of m-THPC showed a significantly higher PDT effect than those extracted after 2 h, whereas llularly . Similarllularly . MenezesEx-vivo investigations suggest that the early effect at 2 h is probably due to a vascular mechanism possibly linked to high plasma concentrations, with a more direct cell kill mechanism being involved at 24 h. This latter peak correlated with maximum tumour concentrations of m-THPC.The biological half-life of m-THPC in the BDIX rat model, as determined using a noncompartmental pharmacokinetic approach, is 22.6 h. This is much lower than that for a Photofrin equivalent that has a half-life of 80 h in the same model. The photodynamic effect of m-THPC in the BDIX rat fibrosarcoma shows two peaks of activity in the 96 h study.


The aim of this study was to elucidate the prognostic impact of putative cancer stem cell markers CD133, CD166, CD44s, EpCAM, and aldehyde dehydrogenase-1 (ALDH1) in colorectal cancer.A tissue microarray of 1420 primary colorectal cancers and 57 normal mucosa samples was immunostained for CD133, CD166, CD44s, EpCAM, and ALDH1 in addition to 101 corresponding whole tissue sections. Invasive potential of three colorectal cancer cell lines was tested.P<0.001). Loss of membranous CD166 and CD44s were linked to higher pT , pN , an infiltrating growth pattern , and worse survival  in univariate analysis only. Loss of membranous EpCAM expression was also linked to higher pN (P=0.023) and infiltrating growth pattern (P=0.005). The CD44s, CD166, and EpCAM expression were lost towards the invasive front. The CD44−/CD166− cells from three colorectal cancer cell lines exhibited significantly higher invasive potential in vitro than their positive counterparts.Differences between normal tissue and cancer were observed for all markers (Loss, rather than overexpression, of membranous CD44s, CD166, and EpCAM is linked to tumour progression. This supports the notion that the membranous evaluation of these proteins assessed by immunohistochemistry may be representative of their cell adhesion rather than their intra-cellular functions. Increasing evidence suggests that, similar to normal tissues, cancers might also be hierarchically organised. Only a minority of tumour cells, endowed with stem cell-like features, and thus termed cancer stem cells (CSCs), might be responsible for tumour initiation and maintenance . NotablyPutative CSC populations have been identified in several types of solid tumours, on the basis of the expression of specific markers and on functional stem cell-like properties, including high clonogenicity, differentiation capacity, spheroid formation, and, critically, the ability to reproduce the original tumour on transplantation in immunodeficient mice .Phenotypic characterisation of CSCs derived from colorectal cancers is still debated. While initial works identified CD133 molecule as a reliable CSC marker in primary human colorectal cancers , distant metastasis (n=489) and adjuvant therapy (n=478) was available. Patient characteristics are summarised in Archival paraffin-embedded material from 1420 patients with primary, pre-operatively untreated colorectal cancer were retrieved from multiple centres including the Institute of Pathology, University Hospital of Basel, Switzerland; the Institute of Clinical Pathology, Basel Switzerland; and the Institute of Pathology, Stadtspital Triemli, Zürich, Switzerland. All histopathological information was systematically re-reviewed from the corresponding hematoxylin and eosin slides including pT classification, pN classification, tumour grade, histological subtype, and the presence of vascular invasion. Tumour border configuration was diagnosed according to Tumour specimens from all 1420 patients as well as 57 samples of normal colonic mucosa were included on a previously described tissue microarray . For invasion assays, cells were stained with APC-conjugated anti-CD44s and PE-conjugated anti-CD166 antibodies , and CD44+/CD166+ or CD44−/CD166− cell subsets were sorted by flow cytometry. Dead cells were excluded by DAPI staining. Purity of sorted cells was ⩾97%. Unsorted tumour cells or sorted subsets were tested for invasiveness in a chemoinvasion assay . Medium containing 5% FCS was seeded in the lower chambers and the cells were incubated at 37°C for 20 h. Inserts were then removed and numbers of cells migrated into the lower chambers were quantified by CyQUANT Cell Proliferation Assay Kit . Percentages of cell invasion were calculated according to the following formula: (relative fluorescent units (RFU) of cells invaded through matrigel-coated membranes/mean RFU of cells migrated through uncoated membranes) × 100.The colorectal cancer cell lines LS180, SW480, and Colo205 were cultured in RPMI 1640 medium supplemented, with GlutaMAX, MEM NEAA, 10 mon assay . Brieflyχ2-tests were carried out for categorical end points. The product-limit method and log-rank or Wilcoxon tests were used to assess differences in survival time. The 5-year survival rates and 95% confidence intervals (CI) were obtained. For Cox multiple regression analysis, the assumption of proportional hazards was verified before each analysis. Patients with missing clinicopathological data or with non-evaluable immunohistochemistry were excluded from the analysis. Hazard ratios (HR) and 95% CI were obtained to assess the prognostic effect of each protein marker on outcome. All tests were two-sided and P-values were considered statistically significant with P<0.05.The P<0.001). Similarly, for CD44s and CD166, expression in normal tissue was detectable in 4.3 and 41.3% of cells on average, respectively, as compared with 33.1 and 64.4% in tumour (P<0.001). Percentages of EpCAM-expressing cells were slightly decreased in tumour compared with normal tissue, with a mean of 95.8 and 100% of positive cells, respectively (P<0.001). Similar results were noted for ALDH1, with an average expression of 15.1% in normal tissue as compared with 10.0% in tumour tissue (P<0.001).The mean percentage of cells expressing CD133 was 0.5% in normal tissue and it significantly increased in colorectal tumours  displayed overexpression, whereas the remaining 629 cases (50.5%) showed loss. Neither overexpression nor loss of CD133 was significantly associated with tumour progression or survival time.P=0.002), lymph node metastasis (P=0.004), an infiltrating tumour border configuration (P<0.001), and worse overall survival compared with patients with CD166 overexpression , lymph node involvement (P=0.002), the presence of vascular invasion (P=0.048), left-sidedness (P=0.008), and an infiltrating tumour border (P=0.002). Furthermore, in 467 cases, for which information on local recurrence was available, a trend between loss of CD44s and local recurrence (P=0.052) was also observed. The 5-year survival rate for patients with loss of membranous CD44s was 53.4% (95% CI: 49–58%), considerably poorer as compared with those with CD44s overexpression, which was 59.3% (95% CI: 55–63% P=0.019)  . HoweverP=0.023) and an infiltrating tumour margin (P=0.005), as well as with a trend towards higher tumour grade (P=0.088) and the presence of vascular invasion (P=0.056). Furthermore, in 489 cases, a decreased EpCAM expression showed a trend towards the presence of distant metastasis (P=0.084). No association of EpCAM expression with survival time was noted either in univariate analysis or after adjusting for the effect of the tumour border configuration.Membranous EpCAM expression was evaluable in 1278 cases of which 1145 (89.6%) showed a diffuse staining in 100% of tumour cells . A totalP=0.025) but not to differences in survival time.Of the 1287 tumours evaluable for cytoplasmic ALDH1 expression, 987 (76.7%) had 0% immunoreactive tumour cells. Overexpression of ALDH1 expression, defined on the basis of ROC analysis as >25% of positive cells, which was observed in the remaining 300 cases (23.3%), was related to higher tumour grade (P<0.001)  . DespiteP<0.001) and 56 of 89 (62.9% P=0.008) cases, respectively, thus confirming our tissue microarray findings. Next, we compared protein expression in the tumour centre with that at the tumour invasive front. Our results indeed showed a heterogeneous expression pattern for both CD166 and CD44s. In particular, decreased expression from the tumour centre to the tumour border was observed in 51 of 100 cases for CD166 and in 47 of 99 cases for CD44s. Importantly, 80.4% (P<0.001) of cases showing reduced expression of CD166 and 78% (P<0.001) of those showing reduced expression of CD44s had an infiltrating border configuration, thus, confirming a positive association between loss of CD166 or CD44s and tumour spreading. Third, we evaluated whether this expression pattern resulted in a poorer effect on survival. Patients with tumours showing decreased levels of CD166 or CD44s expression towards the invasive tumour front when compared with the tumour centre had a significantly more adverse outcome compared with those with no loss of either marker (P=0.006) (P=0.017) and 1.73 , respectively, indicating that the poorer outcome in patients with an expression pattern showing a loss of CD166 and CD44s expression towards the invasive front, although highly linked to tumour growth pattern, may be independent of this histological parameter. Thus, despite the possible heterogeneity between expression levels in the tumour centre and tumour front, a diminished expression of CD166 and CD44s seemed to be consistently associated with tumour progression and unfavourable clinical outcome.The above findings suggested a positive association between loss of CD166 and CD44s molecules and more unfavourable prognosis. In view of the fact that surface molecules might be heterogeneously expressed within colorectal cancer tissues (P=0.006) . Notablyin vitro’ model, we investigated the invasive potential of CD44+/CD166+ or CD44−/CD166− cells derived from the human colorectal cancer cell lines, LS180, SW480, and Colo205. All three cell lines displayed a heterogeneous surface expression of CD44 and CD166  of membranous CD44s and CD166 with aggressive tumour-related features such as more advanced pT stage, pN stage, vascular invasion, and an infiltrating tumour growth pattern. In addition, we document a poorer survival time with loss of membranous expression of both these protein markers in univariate but not multivariable survival time analysis, indicating that the poor prognostic impact of CD44s and CD166 may be secondary to their association with other established prognostic criteria. A similar result has been reported in other tumour types, including ovarian and prostate cancer (We hypothesise that our findings concerning decreased (rather than increased) expression of membranous CD166, CD44s, and EpCAM and their association with features of tumour progression are in large part a result of their cell adhesion function. Loss of cell adhesion is known to be a fundamental mechanism underlying the initiation of the metastatic process  and are vs whole tissue sections), and certainly the choice of cutoff scores for the definition of positive staining or staining intensity. Moreover, the intra-cellular localisation of the evaluated staining (membranous/cytoplasmic) must also be discussed. For example, although EpCAM, similar to CD44, is known for its cell adhesion function , it seems to have versatile roles in signalling, cell migration, proliferation, and differentiation, depending on the microenvironment  (We could not confirm the prognostic value of CD133 or ALDH1 in this study (in vitro analysis using three cell lines, we could show similar findings. Although established cell lines might not fully reproduce the behaviour of primary tumours, our in vitro findings strongly suggest that CD44s and CD166 are of functional importance in limiting tumour cell spreading in surrounding tissues, thus underlining the hypothesis that loss of expression of these markers, rather than their overexpression, is associated with a more aggressive tumour phenotype.A few factors might be envisaged as potential limitations of our study. First, information on local recurrence, distant metastasis, and post-operative therapy was only available for patients treated at one diagnostic centre. However, the lack of independent prognostic effects for our two main CSC markers of interest, namely, CD166 and CD44s, suggest that the absence of complete treatment information may only minimally influence our findings. The results of this study also highlight a heterogeneous expression of CD166 and CD44s throughout the tumour. These findings suggest that using single-punch tissue microarray analysis to investigate these and likely other cell adhesion molecules may be suboptimal. Nonetheless, using two additional and different approaches, namely, analysis of whole tissues sections and per se predictive of poor clinical outcome. Loss of expression of CD166, CD44s, and EpCAM is rather linked to an aggressive tumour phenotype, particularly, to the presence of an infiltrating tumour margin that may implicate these proteins and their loss of membranous expression in events occurring at the invasive tumour front.To our knowledge, this is the first systematic assessment of the prognostic value of CD133, CD166, CD44, EpCAM, and ALDH1 in colorectal tumours evaluated on a large number of cases. Our findings indicate that expression of CSC markers is not


Death appeared to be due to hypoglycaemia and the ability to induce this condition correlated with the differences in malignancy between the sublines; WBP1 (X) and WBP1 (V), the more malignant sublines, inducing hypoglycaemia earlier and more rapidly than the least malignant subline WBP1 (A). Possible mechanisms whereby the tumour produces these effects are discussed.A possible basis for the differences in malignancy between three closely related sublines of the WBP1 ascites tumour of the rat has been studied by examining the biochemical changes in rat sera during tumour growth


In a study of 157 patients with lymphoid malignancy, the phenotype of the tumour cells was correlated with the histological classification of the tumour using the Rappaport and the Kiel classifications. The markers used included E, Fc gamma, Fc micron (IgM) and C3d rosetting, estimation of SIg and CyIg, and tests for the expression of HTLA, Ia and ALL. Repeat biopsy specimens were studied in 23 of these patients. The phenotypic features of lymphoblastic malignancy indicated B-cell, T-cell and ALL-positive null-cell tumours in this group. Immunoblastic lymphomas were predominantly of non-capping B-cell type, but T-cell immunoblastic lymphoma occurred in 2 patients. Immunoblastic lymphomas of receptor-silent cells occur, and are ALL- and HTLA-negative. In the category of diffuse, poorly differentiated lymphocytic lymphomas, most cases are of centroblastic and centrocytic tumour of diffuse type, but pure centrocytic tumours and centroblastic tumours occur. The dominant phenotype in this group is of B cells expressing C3d receptors. Nodular poorly differentiated lymphocytic lymphomas (Rappaport) are classified as centroblastic and centrocytic follicular (Kiel) and most express SIg+ C3d+ phenotype. The frequency of this phenotype appeared the same in both diffuse and nodular poorly differentiated lymphocytic neoplasms. The Rappaport group of diffuse well-differentiated lymphocytic lymphoma includes 2 Kiel categories, malignant lymphoma lymphocytic, and malignant lymphoma lymphoplasmacytoid. Cells of the former tumour were considered to be immature B cells resembling those seen in CLL, and characteristically expressing SIg weakly, with a high frequency of single kappa light chain. Cells of the latter tumour are by contrast mature, and are related to the centroblastic and centrocytic follicular tumour by their histogenesis and phenotypic features. Repeat biopsy examinations indicate that T-cell predominance occurs in the prodromal phase of B-cell-predominant tumours of SIg+ C3d+ phenotype. It is concluded that non-Hodgkin lymphoma can be divided into 2 categories: (1) tumours of immature immunologically incompetent cells of lymphoblastic histology and with phenotypic features akin to T, B and Null-cell ALL, and (2) tumours of differentiated lymphocytes expressing the phenotypic features of B lymphocytes, with maturation arrested at one of several stages of an antigen-dependent immune response.


Lauryl gallate (antioxidant food additive E-312) prevents the formation of dimethylbenzanthracene-induced skin tumours in mice, and kills, selectively, tumoral cells on established tumours. This results in total remission, after topical application of the compound on the tumoral mass, without affecting the surrounding tissue. All experiments involving ‘μg of 7,12-dimethylbenz[a]anthracene (DMBA) in a volume of 20 μl, and 15 days without treatment, a solution of 5 μg of phorbol-12-myristate-13-acetate (PMA) with different doses of LG (Fluka) was applied topically three times per week. The doses of LG were 0 (controls), 2.5, 10 and 50 μg in a volume of 20 μl per application. After 6–7 weeks of treatment, tumours appeared on 90% of the control mice, and the behaviour of LG was evaluated as shown inThirty-two IRC mice were individualised and subdivided into four groups , as shown in μg of DMBA, and after 15 days with no application of compounds, 5 μg of PMA was applied three times a week (6–7 weeks) until the tumour appeared. The mice were subdivided into three different groups , depending on the treatment dose after tumour formation. The volume per application was 20 μl, which was sufficient to cover the whole tumour surface and was applied three times a week for a period of 8 weeks.Twenty-four individualised mice were treated with a single application of 25 μg per application, the effect observed is only partial after 8 weeks (μg (μg (μg) on healthy mice does not produce any effect during the same period of time . However, most of the current strategies are designed to inhibit the ATP binding by specific PTKs which are overexpressed in certain tumours (i.e. imatinib mesylate (formerly STI571) for Bcr-Abl in chronic myeloid leukaemia  without inhibiting serine/threonine kinases  established an acceptable daily intake (ADI) for man of 0.2 mg kg−1 body weight as a sum of propyl, octyl and LG (Also, their simple chemical structures and availability in unlimited amounts at a very low commercial cost are important aspects to consider.


Modelling and simulation of heart valves is a challenging biomechanical problem due to anatomical variability, pulsatile physiological pressure loads and 3D anisotropic material behaviour. Current valvular models based on the finite element method can be divided into: those that do model the interaction between the blood and the valve (fluid–structure interaction or ‘wet’ models) and those that do not .Here an anatomically sized model of the mitral valve has been used to compare the difference between structural and fluid–structure interaction techniques in two separately simulated scenarios: valve closure and a cardiac cycle. Using fluid–structure interaction, the valve has been modelled separately in a straight tubular volume and in a U-shaped ventricular volume, in order to analyse the difference in the coupled fluid and structural dynamics between the two geometries.The results of the structural and fluid–structure interaction models have shown that the stress distribution in the closure simulation is similar in all the models, but the magnitude and closed configuration differ. In the cardiac cycle simulation significant differences in the valvular dynamics were found between the structural and fluid–structure interaction models due to difference in applied pressure loads. Comparison of the fluid domains of the fluid–structure interaction models have shown that the ventricular geometry generates slower fluid velocity with increased vorticity compared to the tubular geometry.In conclusion, structural heart valve models are suitable for simulation of static configurations , but in order to simulate full dynamic behaviour fluid–structure interaction models are required. The mitral valve leaflets form the largest surface area of the four heart valves and are subjected to the highest pressure load during systole, a factor which contributes to the degeneration and dysfunction of a diseased valve. Correct function of the mitral valve requires the synergy of four valvular components: (1) the annulus, (2) the leaflets, (3) the chordae tendineae and (4) the papillary muscles. Valvular dysfunction arises from functional changes in the ultrastructure in any of the components, which can be caused by a range of factors such as infection, variability in the circulatory system or old age The biomechanics of the mitral valve is a complex problem due to the large deformations of the structure, 3D anisotropic non-linear elastic behaviour of the valvular tissue and pulsatile haemodynamic loads during the cardiac cycle. Imaging techniques such as magnetic resonance imaging and echocardiography Models of the mitral valve can be broadly divided into two groups: (1) structural models which only contain the structural domain of the valve and (2) fluid–structure interaction (FSI) models which include both the fluid domain of the blood and the structural domain of the valve. In a ‘dry’, structural model the transvalvular pressure load is applied directly to the valve structure as a boundary condition and has been modelled as uniformly distributed over the surface in all current models. In a ‘wet’, FSI model the two domains are coupled together and corresponding dynamics are solved simultaneously ; the pressure load applied to the valve structure is defined from the coupling applied to the two domains, not the boundary condition applied to the model, and is non-uniform.Numerical studies of the mitral valve have been performed by a number of authors, with each using modelling to examine the role of different valvular aspects; for example the role of the chordae tendineae Hence the objective of this work is to verify how the predicted valve behaviour is affected by the difference in modelling techniques currently used, with particular emphasis on the difference in valvular dynamics between structural and FSI techniques and the influence of the fluid volume on the valve. Using a model of the mitral valve, simulations of both valve closure and the entire cardiac cycle have been performed in “dry”  and “wet” (FSI) scenarios using a commercially available finite element code. As previous FSI studies of the mitral valve have been performed solely in tubular geometries, in this work the effect of the fluid volume on valve dynamics and vice versa is quantified with FSI simulations in both tubular and ventricular-shaped fluid volumes. This has particular implications for hydrodynamic testing of artificial valves, as these are performed in straight fluid chambers as required by current international regulations  have been assumed to be rigid.2The structural and fluid dynamics of the mitral valve and the surrounding blood were simulated using the explicit finite element code LS-DYNA 2.1The different components of the mitral valve were modelled using anatomical measurements and descriptions from the literature The annulus of the model has been defined as a planar D-shaped orifice, which is fixed spatially. The annular proportion and length of the different cusps have been defined from anatomical measurements of excised human valves In this model only the primary chordae tendineae, which are attached to the free margin of the leaflet, have been modelled. The number and location of these chordae have been based on anatomical descriptions 2.1.1−3.The geometry of the valve model was discretised into a finite element mesh of shell elements for the leaflets and beam elements for chordae tendineae. The finite element shells and beams used in this model were Belytschko--Tsay shell elements and Hughes--Liu beam elements respectively. The beam elements have been modelled as cables, which collapse under compression as they generate no force. The physical properties of the shell and beam elements are listed in Eke was reduced to a value smaller than the maximum of the global kinetic energy −6. The solution was considered independent when no observable difference in the stress pattern was noted and the difference in the value of the peak principal stress was less than 1% between models. The final model consisted of 2688 shell elements and 333 beam elements  over 300 ms was applied and sustained on the structure of the model until the solution converged. These simulations were run using a global nodal damping factor in LS-DYNA that was set to 0.9965, a value which was based on previous experience elements .2.23 for the atrial section and 85 cm3 for the ventricular section was chosen. The fluid volume geometries and the surfaces used to apply the transvalvular pressures are shown in The valve model has been inserted and simulated within: (1) a tube-shaped fluid volume and (2) a ventricular-shaped fluid volume. These fluid volumes are static and rigid, with a fixed volume. The geometry of these fluid volumes represent the left ventricle of the heart only and have been sized on the volumetric measurements of the left ventricle. As the FSI simulations include aspects of systole and diastole, an intermediate volume of 35 cm2.2.1−3. As the fluid model of LS-DYNA is compressible, the bulk modulus must be defined. In an explicit finite element formulation this factor controls the size of the maximum timestep of the simulation; thus, to reduce the required simulation time the value of the the bulk modulus was reduced to 1% of its physical value, following The geometry of the two fluid volumes was discretised into solid LS-DYNA ALE fluid elements, with a mesh designed such that the valvular orifice matched the shape of the valve annulus . The flu−1. The solution was considered converged when the absolute difference in the velocity between subsequent timesteps was of the order of 10−6. Once stabilised, the velocity across the valve plane was compared until the difference between subsequent models was reduced to less than 5% in the region of interest. The final models of the tubular and ventricular volumes consisted of 81,920 and 43,008 solid brick elements respectively, as shown in As with the structural mesh, the element size of the fluid mesh was selected such that the independence of the solution to the mesh was obtained. This was performed by applying a velocity boundary condition to the inlet of each model equal to 1 m s2.3Using the structural and FSI models, the structural and fluid dynamics have been simulated using two different pressure loads: (1) early systolic pressure ramp (ESPR) simulating valve closing and (2) a full cardiac cycle simulating both valve opening and closing see  [12]. Fo2.3.1The transvalvular pressures described in 2.3.2In the FSI simulations, the transvalvular pressure was applied to the boundaries of the fluid models see  and the Under the application of the ventricular pressure ramp  the fluiFluid flow was restricted to flow through the valve by applying a no-slip condition to the nodes in the plane of the valve. The boundaries of the volume, with the exception of the inflow and outflow sections  in the tubular and ventricular models, as viewed through the symmetry plane of the fluid volume, is shown in In the fluid domain, the change of sign in the transvalvular pressure gradient causes the flow to change direction between the D1, S and D2 phases of the pressure curve see . In the z-velocity in the valvular plane in the tubular and ventricular models is listed in t = 257 ms is presented in Following the D1 phase is the S phase, during which the flow direction reverses causing the valve to close. The closure of the valve generates a fluid jet, or dynamic regurgitation, forcing fluid back into the atrial section. The timing and maximum magnitude of the nodal t = 685 ms is presented in t = 257 ms and t = 685 ms) in both tubular and ventricular fluid geometries is listed in After closure of the valve in the S phase, the valve re-opens during the D2 phase. As flow enters the ventricular chamber of the fluid volumes, fluid mixing generates vorticity in the flow. A particle trace following the velocity field of the fluid in the tubular and ventricular models at 44.1The configuration of the valve at closure in the different models is compared in The corresponding distribution of local  maximum of the principal stresses are compared in For closure events only, structural models simulate a similar stress distribution to FSI models, although with a lower magnitude of stress. However structural models do have a clear advantage over FSI in terms of the required simulation time, as shown in 4.2The difference in the nodal velocity of the valve structure, in the anteroposterior section, during opening and closing has been compared in The nodal coordinates and corresponding velocities of the nodes in the anteroposterior section of the valve during valve opening (S phase) of the different models are presented in From comparing the coaptation of the leaflets, subtle differences were found in the two FSI simulations. In the tubular model both leaflets were displaced to the centre of the orifice during closure, which resulted in the posterior leaflet making contact with the centre of the anterior leaflet. With the further application of pressure the point of coaptation then moved towards the anterior leaflet tip. In the ventricular model, the posterior leaflet sustains a smaller displacement and the anterior leaflet contacts the posterior leaflet close to the tip of the anterior leaflet and with the application of further pressure no further displacement occurs.Variation in the GOA in both FSI models during the D1 and D2 phases were found to be marginally smaller than the structural model, with the ventricular model larger than the tubular model see . During In −1Following this first filling phase is the S phase during which the direction of fluid flow reverses and closes the valve. The motion of the valve during closure generates a dynamic regurgitant fluid jet in both FSI models. From measuring the velocity of the fluid nodes lying in the valvular plane of the fluid volumes, the maximum velocity was found to occur at approximately the same time but the magnitude in the tubular model was found to be approximately 50% greater than that of the ventricular model , which cAfter the S phase comes the D2 phase, during which the valve reopens and flow passes from the atrium to the ventricle. As the valve opens a fluid jet forms, generating vorticity in the ventricular chamber of the fluid volume. Due to the faster transvalvular flow, the maximum value of the vorticity was found to be greater in the tubular model than the ventricular model see . HoweverAnalysis of the two fluid volumes used in the FSI simulations has shown that the valvular dynamics, in terms of the nodal configuration at closure and the nodal velocities during opening and closing, are comparable between the FSI models although differences were noted in the coaptation dynamics. The effect on the coupled fluid dynamics in the fluid volumes were found to generate higher velocity flow in the tubular volume during opening and closing, along with differences in the fluid vorticity during reopening.4.3The valvular model used in this work has been useful in analysing the differences between the structural and FSI modelling techniques, but it has limitations such as the uniform thickness and the linear elastic behaviour of the valve model. Although the anterior cusp and the central posterior cusp coapt successfully, the valve does not fully close in the commissural areas. Due to the expensive computational time required, the FSI simulations presented here have only been performed for a single cycle.The fluid volumes used to represent the left atrium and ventricle have been sized using realistic volumetric measurements, but are non-physiological in that the boundaries are modelled as rigid and static; the contractility of the LV chamber and the papillary muscles have been neglected.However the models incorporate aspects which have not been previously examined in heart valve modelling and offer a qualitative description of the valve behaviour under different operative conditions. The same modelling techniques can also be applied to tissue 5Here the difference between ‘dry’ structural models and ‘wet’ fluid–structure interaction models have been presented in two separate simulations, one simulating closure only and the other simulating the full cardiac cycle. From the closure simulation it has been shown that the structural model is similar to the FSI models in terms of the predicted stresses but not the stress magnitude or the valvular configuration. Although there are differences between the models, the numerical complexity of a FSI simulations highlights the suitability of structural simulations for the modelling of static configurations such as closed valves.In the cardiac cycle simulation the coupled dynamics of the structure and the fluid have been examined. It was found that the behaviour of the valve varied significantly between the structural and FSI models due to the uniform pressure distribution in the structural model; the dynamics of the valve in the two fluid volumes were found to be similar, with subtle difference in the coaptation of the valve. Significant differences in the fluid domains of the FSI models were noted, with the tubular model exhibited faster fluid velocity during both opening and closing of the valve and a reduced fluid vorticity when compared to the ventricular model.In conclusion, structural heart valve models are more reliable for simulation of static events, but in order to accurately simulate full dynamic behaviour FSI models are required. This dynamic motion is essential in the simulation of valvular repairs, such as the edge-to-edge technique, where the valvular dynamics are affected by reduction of the effective valvular orifice and restricted motion of the leaflets themselves. Also, as the mitral valve regulates the flow in the heart, FSI models simulating normal and pathological states  are important, as they may allow qualification on the role valve motion has on the flow rate and the effect on the circulatory system.None.


By covalent conjugation of amino acids to m-L-SL, a peptide complex consisting of six m-L-SL-based oligopeptides known as peptichemio (PTC) was developed previously. In the present study, the cytotoxic activity pattern of the different oligopeptides of PTC was investigated in ten human tumour cell lines representing different mechanisms of cytotoxic drug resistance using the fluorometric microculture cytotoxicity assay (FMCA). In the cell line panel, L-prolyl-m-L-sarcolysyl-L-p-fluorophenylalanine (P2) was the most active oligopeptide, showing slightly lower mean IC50 values (2.6 vs 3.9 and 4.1 microg ml(-1)) than Mel and m-L-SL. The other five oligopeptides were less active than Mel. All active oligopeptides showed mechanistic similarity to Mel as judged by the correlation analysis of the cell line panel log IC50 values (R > or = 0.90), although P2 appeared to be less sensitive to GSH-mediated drug resistance. The relative activity of Mel and P2 was found to be related to degree of proliferation, P2 being more active towards low-proliferating cell lines. P2 and Mel were then further characterized in 49 fresh human tumour samples. In these samples P2 was considerably more active than Mel and showed a higher relative solid tumour activity (2.7 to 4.5-fold). However, the correlation of log IC50s between P2 and Mel in patient cells was high (R = 0.79), indicating a similar mechanism of action in this tumour model too. Cross-resistance with other standard drugs was lower for P2 than Mel. The results show that P2 is the most potent component of PTC and demonstrates a favourable activity profile compared with Mel. These data suggest that further investigation of P2 as a potential anti-tumour agent is warranted.