P=0.0336) in these tumour xenografts compared to Detroit 562- and CAL27-grafted cell lines with relatively low VEGF levels that were not sensitive to ZD6126. In contrast, neither IL8 levels nor EGFR expression was linked to the antitumour effects of ZD6126. ZD6126 in combination with gefitinib resulted in a synergistic cytotoxic interaction with greater antitumour effects than gefitinib alone. The synergistic interaction between ZD6126 and gefitinib was corroborated by a significant decrease in CD31 labelling. The present study may serve for future innovative clinical applications, as it suggests that VEGF tumour levels are possible predictors for ZD6126 antitumour efficacy. It also supports the notion of antitumour supra-additivity when combining gefitinib and ZD6126, and identifies neoangiogenesis as the main determinant of this synergistic combination.Current experimental and clinical knowledge supports the optimisation of endothelial cell targeting using a strategy combining anti-EGFR drugs with antivascular agents. The purpose of the present study was to examine the effects of the association of ZD6126, an antivascular microtubule-destabilising agent, with gefitinib and irradiation on the growth of six head and neck human cancer cell lines xenografted in nude mice and to study predictive and molecular factors responsible for antitumour effects. CAL33- and Hep-2-grafted cell lines were the most sensitive to ZD6126 treatment, with VEGF levels significantly higher ( Tumour expression of growth factor receptors of the HER family makes it possible to perform innovative therapeutic targeting as demonstrated by the development of clinically active drugs such as herceptin(®), cetuximab and EGFR tyrosine kinase inhibitors . This abTumour vasculature is a key target in the treatment of solid tumours, particularly of the head and neck . In the Gefitinib and ZD6126 were kindly provided by AstraZeneca . Working solutions were prepared extemporaneously as follows: gefitinib was suspended in 0.9% NaCl, 0.01% Tween-80 and ZD6126 in 0.9% NaCl with pH adjusted to 4.5. For both drugs, the concentrations were adjusted so as to include the daily dose in 0.2 ml of drug suspension. Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin and glutamine were purchased from Whittaker . Foetal bovine serum (FBS) was obtained from Dutscher .in vitro sensitivities to gefitinib or RT and p53 status, as summarised in Six different human head and neck cancer cell lines: CAL27, CAL33, CAL60 and CAL166 originated from our institution (the Centre Antoine Lacassagne); Detroit 562 and Hep-2 were obtained from the American Type Culture Collection . These cell lines exhibit variable EGFR levels , 2 mM glutamic acid, 50 000 U l−1 penicillin and 80 μM streptomycin in a humidified incubator  at 37°C in an atmosphere containing 8% CO2. Batches of 15 × 106 cells were frozen in FBS supplemented with 5% DMSO (v v−1) in advance for injection into mice. Shortly before injection, cells were thawed and suspended in Ringer lactate.The cell lines were maintained as monolayer cultures in DMEM supplemented with 10% FBS .Animal experiments were performed in accordance with the regulations of the institutional ethical commission and of the United Kingdom Co-ordinating Committee on Cancer Research guidelines .Tumour length and width were measured weekly using a caliper. Tumour volume was calculated as M Tris-HCl buffer (pH 7.4), containing 1 mM EDTA, 0.5 mM dithiothreitol and 10 mM sodium molybdate. The homogenates were centrifuged for 1 h at 105 000 g (+4°C) and the supernatants (cytosols) were used for protein determination by immunoblotting. Total protein content was measured using the bicinchoninic acid assay.Frozen tumours were pulverised in a liquid nitrogen-cooled Thermovac pulveriser. The resulting powders were homogenised in 10 volumes of a 10 m125I-EGF binding method followed by Scatchard-plot analysis. These factors were chosen because VEGF is the main growth factor for endothelial cells; IL8 is a proangiogenic factor  with a proven role in different tumour types and EGFR as it is the target of gefitinib.VEGF and IL8 were determined by ELISA using DVE 00 and D8050, respectively, from Quantikine ; EGFR was determined by the The microvessel marker CD31  and the proliferation marker Ki67  were determined by MTA on treated tumours obtained after animal killing.3. The differential expression of molecular factors potentially related to ZD6126 sensitivity such as VEGF, IL8 (by ELISA) and EGFR (by ligand-binding assay) was measured, at the end of the observation period, in untreated tumours (tumour volume between 72 and 2344 mm3) after animal killing.Tumour volume was monitored once a week between 3 and 8 weeks depending on the cell line; the experiments were stopped when tumours reached 1 cm3; animals were treated once a week for 3 weeks, with freshly prepared ZD6126 (200 mg kg−1)  were examined for the impact on tumour growth and tumour angiogenesis (CD31) and on molecular factors indicative of proliferation (Ki67). CD31 and Ki67 expressions were determined in MTAs by immunohistochemistry (3) were treated every week with vehicle alone (controls), ZD6126 , gefitinib  and RT . The dose of gefitinib was chosen according to previous data in tumour xenografts in mice  was evaluated for tumour growth inhibition. The sequence between gefitinib and RT was already established by us from tumour experiments with the two agents applied to the same tumour cell line xenograft . The differences between cell lines for molecular factors  were examined using the Mann–Whitney test.The fastest growing xenografts were CAL166 and CAL33 with a tumour volume doubling time of 7 and 10 days, respectively, followed by Detroit 562 (tumour volume doubling time of 15 days) . The othThere was a marked variation in VEGF and IL8 levels between the six xenograft models examined. The variability in EGFR expression was smaller between the studied cell lines .The tumour volume did not influence VEGF secretion ; tumoursP=0.0336) in these tumour xenografts as compared to Detroit 562 and CAL27 xenografts for which ZD6126 increased the tumour growth (ZD6126 on its own exhibited relatively modest tumour growth inhibition. There were marked disparities in the antitumour activity of ZD6126 between the different xenografts . Of noter growth . NeitherP<0.01) (At the end of the experiment (day 33), CAL33 tumours were analysed for CD31 and Ki67 staining. Single-drug treatments with either gefitinib or ZD6126 had a modest effect on CD31 tumour labelling as compared to controls without drug . CD31 tuP<0.01) . There wP<0.01) . In contP<0.01) . SimilarN-acetyl colchinol which has been shown to induce pronounced but reversible changes in immature endothelial cell morphology and to have marked vascular effects and antitumour activity in preclinical models (Tumour neoangiogenesis is a complex process that involves multiple and interrelated steps dependent on positive and negative regulatory growth factors , the VEGl models . Head anl models , 2004 anThere is evidence that ZD6126 is active in various preclinical tumour models with variable antitumour efficacy (The second part of this study was designed in a way similar to the work recently published by Previous experimental studies showed potential beneficial antitumour effects when combining ZD6126 with RT  that shoOverall, the present study may contribute to future innovative clinical applications and suggests that the VEGF tumour level is a possible predictor for ZD6126 antitumour efficacy, that it strengthens the notion of antitumour supra-additive effects when combining gefitinib and ZD6126, identifies neoangiogenesis as the main impact of this synergistic combination and gives no firm support to the benefit of adding RT to the drug association.


The effects of adriamycin (Adr) on the solid-tumour model, hepatoma 3924A, and on critical organs of the host, were determined at intervals after single injections of 60 mg/m2 of the agent. A reduced rate of tumour growth was evident 4 days after treatment, continued to Day 11, and then returned to rates comparable to control values. On Day 11 tumour volumes of treated animals were 38% of control. During the period of reduced growth, 3H-TdR incorporation into tumour DNA and percentage labelled tumour cells were less than control values. DNA concentration in tumour was not affected by drug treatment, which differs from observations made in other studies employing 5-fluorouracil (FU). No evidence of significantly increased necrosis or fibrosis of the tumour was found after Adr. The Adr treatment caused loss of 60% of the tibial marrow by Day 4, as measured by total DNA content. Marrow DNA recovered to control levels between Days 7 and 11. Incorporation of 3H-TdR into heart DNA was reduced more than 40% during the first week after Adr treatment; enhanced incorporation was observed on Day 11, and control levels were attained by Day 17. No significant pathological evidence of cardiac toxicity was found 2-21 days after Adr but degeneration of myocardial cells and oedema was prominent at 14 weeks.


C3Hf/Dp mice were given a single i.p. injection of 50, 25 or 5 mug/g N-Nitroso-N-Methylurea (NMU) at either 1 or 70 days of age or 50 mug/g at 21 days of age. They were observed until death or until 120 weeks of age. The two highest doses of NMU produced tumours in a wide spectrum of organs, including the thymus, forestomach, lung, liver , kidneys, ovaries and orbital glands. The only two tumour types which appeared to be closely related to the occurrence of death were thymic lymphomata (most of which were found in mice dying before 40 weeks after treatment) and carcinomata of the forestomach. Lifetime analyses are presented concerning the occurrence of these two tumour types as well as the occurrence of any tumour after 40 weeks of age or since treatment. Incidences of thymic lymphomata were 67.6%, 39.0% and 21.2% in mice receiving 50 mug/g NMU at 1, 21 and 70 days respectively and 17.1% in mice receiving 25 mug/g at 1 day. In the other groups the incidence of thymic lymphomata was zero or negligible. The rate of progression of thymic lymphomata until death was related to both earliness of treatment and dose. On the contrary, incidences and progression of carcinomata of the forestomach were unrelated to age at treatment. Since breakdown of NMU is very rapid and does not require enzymes, these results are considered as evidence that host-tumour interaction differs from organ to organ. No excess of tumours over the controls was found in mice receiving 5 mug/g either at 1 or 70 days of age.


Female strangulation in South Africa occurs in a context of pervasive and often extreme violence perpetrated against women, and therefore represents a major public health, social and human rights concern. South African studies that provide accurate descriptions of the occurrence of strangulation incidents among female homicide victims are limited. The current study describes the extent, distribution and patterns of homicidal strangulation of women in the four largest South African metropolitan centres, Tshwane/Pretoria, Johannesburg, Cape Town and Ethekwini/Durban.The study is a register-based cross sectional investigation of female homicidal strangulation, as reported in the National Injury Mortality Surveillance System for the four cities, for the period 2001 to 2005. Crude, unadjusted female strangulation rates for age and population group, and proportions of strangulation across specific circumstances of occurrence were compiled for each year and aggregated in some cases.This study reports that female homicidal strangulation in urban South Africa ranges from 1.71/100 000 to 0.70/100 000. Rates have generally declined in all the cities, except Cape Town. The highest rates were reported in the over 60 and the 20 to 39 year old populations, and amongst women of mixed descent. Most strangulations occurred from the early morning hours and across typical working hours in Johannesburg and Durban, and to a lesser extent in Cape Town. Occurrences across Johannesburg, Durban and Pretoria were distributed across the days of the week; an exception was Cape Town, which reported the highest rates over the weekend. Cape Town also reported distinctly high blood alcohol content levels of strangulation victims. The seasonal variation in strangulation deaths suggested a pattern of occurrence generally spanning the period from end-winter to summer. Across cities, the predominant crime scene was linked to the domestic context, suggesting that perpetration was by an intimate partner or acquaintance.The study contributes to an emerging gendered homicide risk profile for a country with one of the highest homicide rates in the world. The results support the call for the development of evidence-based and gender-specific initiatives to especially address the forms of violence that instigate fatalities. Gender-based violence persists as a global public health problem. In 2000, there were an estimated 119 000 female homicides worldwide, for an overall age-adjusted rate of 8.8 per 100 000 population [The international body of literature on female homicide victimisation reveals a proliferation of studies during the last decade that seek to explore the patterns and correlates of homicide victimisation among women. This field of research is dominated by studies that have investigated: (1) female homicide victimisation patterns of occurrence within and between countries -6; (2) tStrangulation is considered to be a form of mechanical asphyxia . The mecAlmost all attempted or completed homicides by strangulation involve either ligature strangulation (strangulation with a cord-like object) or manual strangulation  . LigaturMany of the findings reported here are based on small numbers of cases that limit the conclusions that can be drawn, and are essentially descriptive in nature and therefore do not take into account socio-political-cultural concepts considered to be central to the understanding of gender-based violence. The results are nonetheless instructive in the preliminary identification of risk profiles for homicidal asphyxia and the development of implied interventions.Despite the burgeoning scholarship on the subjects of female homicide victimisation and of asphyxial homicide, the research evidence reviewed highlights that systematic investigations directed specifically at strangulation in the context of female homicide victimisation remain meagre. The near absence of such research in South Africa, against the backdrop of findings emerging from the single documented national study of female homicide in South Africa , as wellThe current study explores the distribution and patterns of homicidal strangulation of women in the four largest South African metropolitan centres, Tshwane/Pretoria, Johannesburg, Cape Town and Ethekwini/Durban, for the period 2001 to 2005. These cities register amongst the highest rates of homicide in the country . The fol1. What is the incidence of female strangulation in four South African cities?2. What is the distribution of female strangulation across age and population groups?3. What are the typical circumstances of occurrence of female strangulation?The study is a register-based cross sectional study of female homicidal strangulation, as reported in the National Injury Mortality Surveillance System (NIMSS) for four South African cities over the period 2001 to 2005. The study utilised mortality rates for the purpose of comparability of data, a departure from retrospective analyses of forensic autopsies and related hospital records ,27,30.The current study investigated strangulation fatality data recorded by the NIMSS. The NIMSS produces and disseminates descriptive epidemiological information on deaths due to non-natural causes that, in terms of South African legislation, are subject to medico-legal investigation. The NIMSS is a collaboration between the South African Department of Health (DoH) and the Crime, Violence and Injury Lead Programme (CVI); the latter is co-directed by the Medical Research Council (MRC) and the University of South Africa (UNISA). Information for this system is collected by the police and forensic pathologists at each mortuary, and captured onsite into a computerised database by clerks and secretarial staff. The NIMSS records 21 items of information, detailing, where available, the deceased person's age, sex, population group, province, town and suburb of injury, scene of injury, apparent manner and circumstances  of death . The NIMAt the time of the study, the South African Census 2001 description was the most comprehensive database available for the four cities. The Census 2001 database was used as a base for city denominator extrapolations for 2002 to 2005, based on the parameters and city growth rates specified by a population model developed by the Actuarial Society of South Africa ,41. The A descriptive analysis of all female homicidal strangulations was computed for each city: by age category ; and by population group . The selected age categories were consistent with those specified for injury analyses by the WHO. Crude, unadjusted mortality rates were calculated for each city for the 2001 to 2005 period using SPSS, by relating the number of cases for each year from 2001 to 2005 to the city population estimated for each year and expressed per 100 000 per annum. The proportions of female strangulation occurring over the 5 year period were computed for each city for time (6 categories); day (7 categories); month of death (twelve categories); scene of injury (17 categories); and for blood alcohol content (BAC) (3 categories).Table The overall strangulation rate was at its highest in Durban in 2003, with 1.36 deaths per 100 000 population, and at its lowest in Pretoria in 2005, at 0.57 deaths per 100 000. There was a general decline in strangulation rates between 2001 and 2005 in Pretoria (from 0.9 to 0.57 deaths/100 000) and in Johannesburg (0.96 to 0.76/100 000). In Cape Town, the homicidal strangulation rate was relatively stable, marginally increasing from 1 to 1.1 deaths/100 000 across the five years. The overall strangulation rate varied most in Durban, from 1.1/100 000 in 2001, peaking at 1.36/100 000 in 2003, and thereafter declining to 0.62/100 000 in 2005.The four major causes of homicidal death were consistently ranked across the four cities in the following order: firearm discharge, sharp objects, blunt objects and thereafter strangulation . Of thesTable apartheid, had impacted on the lives of various groups of South Africans, and still do. Population group affiliation was available for 98.75% of victims. Coloured females reported the highest rates of occurrence in Pretoria , Durban  and Cape Town . This is followed by high rates of black female strangulations in Cape Town  and Durban , and by high rates amongst white women in Cape Town  and Durban . There were no occurrences amongst Asians in Cape Town and Pretoria where the population there constitutes a small minority [Table minority ; rates oTable Figure Figure Across all four cities, the crime scene tends to be within the confines of the home . Other crime scenes included open beach or land, where 17.7% of cases occurred in Durban; residential institutes, where 14–14.6% of cases occurred in Johannesburg and Pretoria; and the road or highway, where for Cape Town 5.8% of cases occurred.Table This study reports that female homicidal strangulation in urban South Africa ranges from 1.71/100 000 in Durban (2003) to 0.70/100 000 in Johannesburg (2003). These rates, especially the former, are high compared to the few reported for other settings, such as the combined overall strangulation rates  of 0.17/100 000 for Jordan and the 1.1/100 000 reported for the United States ,42. The Homicidal strangulation is more prominent in females than males in the metropolitan centres of the country, unlike the higher male rates observed for the other major external causes of urban homicide death, such as firearm and sharp force injury . StranguFemale homicidal strangulation has since 2001 generally declined in two of the cities, Pretoria and Johannesburg. Durban, despite a more erratic pattern of occurrence, also reported an overall decrease of female strangulation. This trend echoes that for the overall homicide rates across the four cities. This decrease has occurred in the context of supportive national legislation , the enhancement of the criminal justice system , and other social and health initiatives . DespiteCape Town is the single exception to this declining trend. The Western Cape Province  also reported the highest homicide rate and amongst the highest number of reported cases of rape for this period . These fThree cities reported the highest rates of strangulation deaths amongst their older populations. Elderly female victims have been reported to be at higher risk for strangulation due to their physical vulnerability and resulting inability to offer much resistance during an attack ,28. The This study reported the highest rates of strangulation amongst coloured women, consistent with the indications of elevated intimate femicide rates amongst coloured females . In thisMost strangulations occurred from the early morning hours and across the typical working day in Johannesburg and Durban, and to a lesser extent in Cape Town. Pretoria occurrences clustered in the early morning hours until midday. These periods indicate the times of greatest vulnerability for strangulation attacks. These attacks may be directed especially at unemployed, home-based women and occur at a time of day when the majority of the population is at work or school, with consequently less immediate protection and safety. Cape Town, unlike the other cities, reported the highest rates over the weekend, consistent with high alcohol consumption over recreational periods. The seasonal variation in strangulation deaths suggests a pattern of occurrence generally spanning the period from end-winter to summer. Increasing day length has been associated with a rise in aggression in young males, and increasing evening warmth and daylight with increased consumption of alcohol in bars and streets, thereby resulting in a higher probability of inebriated encounters compared with other times of the year . Across The study is the first of its kind in South Africa and contributes to an emerging gendered homicide risk profile for a country with one of the highest homicide rates in the world. The data is based on a well-established mandatory mortality surveillance system, which provides reliable forensic data for the cities. However, complete information on both demographics and the circumstances of cases of death are not always possible due to forensic investigative limitations. There is for example an absence of information on the victim-perpetrator relationship, which would be the focus for further South African studies on the subject. The study provides a description of strangulation victims and the circumstances of occurrence, important for the generation of overall policy and prevention guidelines. This data also lends itself to future multivariate pattern analysis, which will provide further descriptions of the scenarios of occurrence and thus more detailed prevention targets.In urban South Africa, fatal violence in females is clearly distinct from fatal violence in men. Accordingly, current prevention initiatives that address gender-based violence need to be strengthened, and supplementary evidence-based and gender-specific initiatives need to be developed to especially address the forms of violence that instigate fatalities. Dedicated policing teams; stronger legislation aimed at protecting women; universal screening for strangulation injury in women assessed to be victims of intimate partner violence; community-level substance abuse and domestic violence prevention programmes; and accessible and affordable support services and networks are some of the critical prevention initiatives that require development and support at both the local and national levels. The prevention of female homicidal strangulation is dependent on the generation of frequent, timely, comprehensive and accurate data on victims, perpetrators and the precipitating circumstances. In particular, homicidal strangulation in women needs to be further investigated in relation to intimate partner violence, as well as sex crimes. Importantly, research is required on the experiences of women who survive non-lethal strangulation to further identify risk factors. The prevention of female homicidal strangulation in South Africa will ultimately be strengthened by the creation of a social milieu that promotes equity, safety, health and human rights, and justice.The authors declare that they have no competing interests.This study was devised by SS and AVN. NA analysed the data. All authors made contributions to interpretation of the data. SS and AVN drafted the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Local immunoregulation mediated by mononuclear tumour-infiltrating cells is considered of importance for tumour progression of colorectal cancer, although the balance between immunosuppressor and cytotoxic activities is unclear. Colorectal cancers from 26 patients were investigated using a panel of monoclonal antibodies in order to identify subsets of mononuclear inflammatory cells and to study their pattern of distribution in relation to tumour stage and cytotoxic immune reactivity against the tumour. In all but five tumours, mononuclear cells, lymphocytes or monocytes were present in fairly large numbers, particularly in the stroma. The infiltration of CD4+ mononuclear cells predominated over the CD8+ subset. Infiltration near the tumour cells was found in four cancers only. Stromal infiltration of CD11c+ macrophages was found in all but eight tumours. Small regressive areas, in which the histological architecture of the tumours was broken down, were found in 17 tumours with intense or moderate infiltration by CD4+ lymphocytes or CD11c+ macrophages. Probably this destruction of tumour tissue was caused by cytotoxic activity of the tumour-infiltrating mononuclear cells. In Dukes' class A and B tumours, CD4+ lymphocytes predominated over CD4+ cells with macrophage morphology, but the latter were increasingly found in Dukes' class C and D disease. The occurrence of MHC II-positive macrophages and lymphocytes in different Dukes' classes was similar to that of CD4+ cells. In contrast to this, CD11c+ and CD11a+ cells were more frequent in Dukes' A and B class tumours compared with Dukes' C and D. Four out of nine tumours of the latter stages showed a poor inflammatory reaction. The interpretation of our results is that the subsets of tumour-infiltrating mononuclear cells change with advancing Dukes' class and that the local immune control is gradually broken down in progressive tumour growth, even if some cytotoxic activity is still present.


As reported herein and by others, brain-specific angiogenesis inhibitor 1 expression is present in some normal tissues, but is reduced or lost in tumour tissues. Such data and its particular structure prompted the hypothesis that brain-specific angiogenesis inhibitor 1 may act as a mediator in the local angiogenesis balance. We herein demonstrate that brain-specific angiogenesis inhibitor 1 over-expression suppresses tumour angiogenesis, delaying significantly the human tumour growth in immunodeficient mice. The inhibitory effect of brain-specific angiogenesis inhibitor 1 was documented using our intravital microscopy system, strongly implicating brain-specific angiogenesis inhibitor 1 as a mediator in the control of tumour angiogenesis. In contrast, in vitro tumour cell proliferation was not inhibited by brain-specific angiogenesis inhibitor 1 transfection, whereas some level of cytotoxicity was assessed for endothelial cells. Immunohistochemical analysis of tumour samples confirmed a reduction in the microvessel density index in brain-specific angiogenesis inhibitor 1-overexpressing tumours. At messenger level, moderate changes could be detected, involving the down-regulation of vascular endothelial growth factor and collagenase-1 expression. Furthermore, brain-specific angiogenesis inhibitor 1 expression that was lost in a selection of human cancer cell lines could be restored by wild-type p53 adenoviral transfection. Brain-specific angiogenesis inhibitor 1 should be considered for gene therapy and development of efficient drugs based on endogenous antiangiogenic molecules.The British Journal of Cancer (2002) 86, 490–496. DOI: 10.1038/sj/bjc/6600067www.bjcancer.comThe Cancer Research Campaign© 2002  Subcutaneous and skin chamber growth of wild-type and transfected tumour cells were observed and measured. We have analyzed visually the early human tumour angiogenesis with our transparent chamber model in immunodeficient mice. In short, this is the first report detailing the inhibitory effect of BAI1 on a human tumour as applied by gene therapy.Considering all the above-mentioned reports, in an attempt to extend the study of the BAI1 effects on human tumours, we checked the expression of this gene in pancreatic and colon cancer. In addition, we have investigated the interrelation between the presence of BAI1 and human pancreatic tumour development, associated very frequently with BAI1 we used six well-known human pancreatic adenocarcinoma cell lines , three cell lines established in our department  of 10 for 36 h, as described  6–8-weeks-old were used in all experiments, and kept in pathogen free conditions. Mice bearing dorsal skinfold chambers were housed individually. This study was approved by the Ethical Committee of Tohoku University Graduate School of Medicine. We observed the United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR) guidelines for the welfare of animals in experimental neoplasia.BAI1 expression on Panc-1 cells and on HUVECs, we performed MTT assays. One thousand tumour cells were plated in a 96-well plate and infected for 36 h with adenoviral vector encoding BAI1 using multiplicities of infection from 0–200 (16 wells for each of the MOI used). Media was changed after 36 h, and 5 days after infection, 10 μl of sterile tetrazolium salt, MTT  was added to the wells and incubated for 4 h at 37°C. Finally, 100 μl of 10% SDS were added, and after incubation at 37°C overnight, the plate was read at 490 nm. The in vitro growth of 2×105 of the Panc-1 transfectants (MOI 10) used for in vivo experiments was compared to wild-type cells by crystal violet (1%) staining at day 6.In order to evaluate the cytotoxic effects of 6 cells, n=5) were assessed by measuring the tumour volume using a calliper. Tumour volumes were estimated by the following formula: V=D×2d×0.4 .Subcutaneous growth curves in NK-depleted SCID mice of Panc-1 wild-type, Panc-1/BAI1, and Panc-1/LacZ  was observed biweekly for 24 days using a Optiphoto 066 microscope , a CCD camera  attached to the microscope, and the images were recorded on a video tape recorder . The recorded data were analyzed off-line.Transparent chamber and 6 tumour cells using RNeasy Mini Kit , then 10 μg aliquots were reversed-transcribed using a RNease H Minus RT , and 1 μg of the resultant cDNA was amplified. Basic FGF, VEGF, MMP-1, ETS-1, and uPA were also amplified. The amplification of MSH2 was used as internal control. The specific amplification conditions are shown in Table 1The expression of BAI1 in normal and tumour pancreatic tissue, and in pancreatic and colon cancer cell lines was assessed by RT–PCR. Total cellular RNA was extracted from a normal and 14 tumour pancreatic tissue samples, and from 1×10n=5) were stained, and capillaries (diameter <10 μm) were counted in 10 high magnification fields per section.Rabbit anti-asialo GM1 antibody  was administered to SCID mice twice a week in order to block the non-specific immunity conferred by the NK cells. Standard haematoxylin and eosin staining was performed on 3–5 μm sections from paraffin-embedded tumour samples. Immunostaining of endothelial cells in tumour tissue was performed using rat anti-mouse PECAM-1 (CD31) antibody , as described . The expression in tumour cells of some of these factors such as basic FGF, ETS-1, and uPA appeared not to be affected by transfer of either the BAI1 or LacZ gene (not shown). However, BAI1 induced a decrease in VEGF level, as well as of that of interstitial collagenase 1 (MMP-1), the only metalloproteinase expressed in Panc-1 cells. This was not due to reporter gene transfection, as indicated by simple and duplex RT–PCR for MMP-1 and MSH2  used for in vivo studies (not shown). Moreover, beta-galactosidase staining of LacZ transfectants (MOI 10) confirmed a very high infection rate  of the tumour cells by the adenoviral-mediated transfer. Although no significant cytotoxic effect of BAI1 on tumour cells was observed by MTT assays, an antiproliferative effect after transfection into HUVECs was indicated  implicated in the local angiogenic and proteolitic balance. Moreover, as reported for wild-type p53, BAI1 over-expression inhibited the expression of collagenase-1 (MMP-1), which is known to be a positive regulator of tumour angiogenesis  was found in other cells to be a p53-target gene, subject to p53 repression, mediated in part by associated protein 1 . It appears that the presence of the integrin-recognition motif RGD plays an important role in antiangiogenesis mechanisms and therefore we are currently investigating the effects of the transfer of BAI1 with a point mutation within the RGD motif.The loss of BAI1 expression plays a role in the tumour angiogenesis-related cascade of events not only in brain-specific tumours, but also in other tumours types. These events are generally associated with the loss of p53 integrity or function. On the other hand, the overexpression of BAI1 inhibited strongly the pancreatic tumour-related angiogenesis. Therefore, since pancreatic carcinoma has a very poor prognosis after surgery, chemotherapy, and radiation therapy, BAI1 should be considered as a relevant candidate for future trials using this type of molecules or peptides derived from them, that would employ targeting of tumour angiogenesis as adjunctive or adjuvant therapy.


Monoclonal antibody TFS-4 has previously been shown to react selectively with human small cell lung cancer (SCLC). We evaluated the use of 131I-labelled TFS-4 for the treatment of established human SCLC transplanted in nude mice. The specific accumulation of the antibody in the transplanted tumour was recorded by both scintigraphic and biodistribution studies. Administration of 200 microCi 131I-labelled TFS-4 inhibited tumour growth when compared with the same radiation dose of the control monoclonal antibody. The therapeutic effect was dose-dependent and complete disappearance of the tumour was observed transiently in one out of the three animals following the administration of 500 microCi 131I-labelled TFS-4.


The spleens of rats bearing methylcholanthrene induced sarcomas are enlarged. This applies both to primary and to isotransplanted tumours. The spleen enlarges with increasing tumour size.Splenomegaly is induced by skin homograft rejection, but spleen sizes do not reach those seen during tumour growth, even when the animals are chronically exposed to homologous skin. Sensitization of animals with small tumour homografts gives spleen sizes even greater than those of primary tumour growth.Spleen histology in tumour bearing animals is comparable with that of homograft rejection.The relation of splenomegaly to the presence of tumour specific antigen is discussed. The suggestion is advanced that the spleen size during tumour growth is determined by the product of reaction to foreign antigen, and of reticuloendothelial (phagocytic) activity.


The anti-tumour action of methylene dimethane sulphonate (MDMS) has been further investigated in relation to its immunosuppressive properties. Following a dose of 10 mg/kg, the proportion of permanent regressions of Yoshida lymphosarcoma transplants is lower in animals treated during the first 5 days of tumour growth. Re-implants on day 28 to those animals in which regression of the tumour had occurred indicated that the immune response to the tumour increases during the first 7 days of tumour growth.Studies of the effect of MDMS on the primary antibody-forming cell response of mice to sheep red cell antigens showed this drug to be an immunosuppressant comparable in strength to x-radiation. MDMS given to rats prior to tumour transplantation also acted as an immunosuppressant in this system resulting in an increased rate of tumour growth. For both responses the maximum immunosuppressive effect was obtained when the interval between drug administration and antigenic challenge was minimal.


HMEC-1 cells were moderately sensitive to continuous exposure to cisplatin, but showed greater sensitivity to bleomycin. Combination of a 5 min drug exposure with electric pulses increased cytotoxicity of cisplatin by ∼10-fold for cisplatin and ∼5000-fold for bleomycin. The electroporation of HMEC-1 cells with bleomycin for a 5 min exposure was ∼250-fold better than a continuous exposure to the drug alone. The results of this study indicate that the anti-tumour action of electrochemotherapy is likely to be due, in part, to the highly sensitive response of vascular endothelial cells. Further studies are necessary to identify the determinants of endothelial response and its relationship to the anti-vascular action of electrochemotherapy in vivo. © 2001 Cancer Research Campaign http://www.bjcancer.comRecent studies have indicated that the antitumour effectiveness of electrochemotherapy, a combination of chemotherapeutic drugs with application of high voltage electric pulses applied to the tumour nodule (electroporation), result in a significant reduction in tumour blood flow and may therefore be mediated by an anti-vascular mechanism. The aim of this study was to evaluate the cytotoxicity of electroporation with bleomycin or cisplatin on cultured human microvascular endothelial cells (HMEC-1). The sensitivity of HMEC-1 cells to a 5 min treatment by electroporation with bleomycin or cisplatin (8 electric pulses, pulse duration 100 μs, frequency 1 Hz, electric field intensity 1400 V cm


On multiple regression analysis, albumin concentrations were independently associated with C-reactive protein  but not percentage hepatic replacement (P=0.34). These results show that hypoalbuminaemia is associated with the presence of a systemic inflammatory response rather than tumour volume in patients with colorectal liver metastases.The relationship between hypoalbuminaemia, tumour volume and C-reactive protein was examined in patients with colorectal liver metastases ( It has long been recognised that, in patients with advanced cancer, the presence of hypoalbuminaemia is associated with poor outcome . In the The nature of the relationship between circulating albumin concentration, tumour burden and the systemic inflammatory response has not been established. However, if the presence of a systemic inflammatory response rather than tumour burden were to determine the development of the hypoalbuminaemia associated with advanced cancer, then it might be expected that there would be a stronger relationship between the presence of hypoalbuminaemia and the systemic inflammatory response compared with that between hypoalbuminaemia and tumour volume.The aim of the present study was to examine the relationship between circulating albumin concentration, the tumour burden and the systemic inflammatory response in patients with colorectal liver metastases.Patients who had undergone resection of a primary colorectal cancer, who then developed liver metastases and who were referred for assessment for liver resection, were considered eligible for study. As part of their assessment, patients underwent dual phase spiral-computed tomography of the liver. In addition a blood sample was taken for measurement of routine liver enzymes, albumin and C-reactive protein. Only those patients who had no evidence of extrahepatic disease were included in the study. None of the patients had recent chemotherapy.The study was approved by the Research Ethics Committee of Glasgow Royal Infirmary.Albumin, C-reactive protein, alkaline phosphatase, aspartate transaminase, alanine transaminase and bilirubin concentrations were assayed using standard methods. Intra- and inter-assay coefficients of variation were better than 5 and 10% respectively.Tumour volume and percentage hepatic replacement (PHR) were measured using a similar approach to that previously described .To assess the accuracy of the CT-derived measurements of tumour volume, the CT measurements of tumour volume were compared with the weight of freshly resected tumour in a separate cohort of nine patients undergoing liver resection. The correlation coefficient between the CT-derived measurements of tumour volume and the weight of the resected tumour was greater than 0.99  was used for analysis.The relationships between albumin, PHR and C-reactive protein were analysed using Pearson correlation coefficients and multiple linear regression analysis with albumin as the dependent variable. PHR and C-reactive protein concentration were logarithmically transformed to normalise their distributions. The appropriateness of the regression model was confirmed by examining residuals graphically. Associations involving liver enzymes and bilirubin were analysed using Spearman's rank correlation coefficient. Comparisons between groups of patients were carried out Fisher's exact test or the Mann-Whitney P=0.034) and C-reactive protein (P=0.006).The characteristics of the patients studied are shown in r=−0.34, P=0.010), albumin and C-reactive protein  and PHR and C-reactive protein . A multiple regression model combining PHR and C-reactive protein as predictor variables  did not significantly improve on a model based on C-reactive protein alone (P<0.001), while the relationship between albumin and PHR adjusted for C-reactive protein was not significant (P=0.34). The relationship between circulating concentrations of albumin and C-reactive protein is shown in Figure 1There were significant correlations between albumin and PHR , aspartate transaminase , alanine transaminase  and bilirubin . Of these liver enzymes and bilirubin, only aspartate transaminase was significantly associated with C-reactive protein  and albumin .There were significant rank correlations between PHR and alkaline phosphatase (It has been recognised for some time that the degree of biochemical upset in patients with advanced cancer depends not only on the tumour burden but also the site of the disease. In advanced colorectal cancer where the tumour is disseminated only to the liver, tumour size is readily assessed by computed tomography and therefore provides a model in which to examine the relationship between hypoalbuminaemia and tumour volume.It is well recognised that albumin concentration is a stage independent prognostic factor in patients with advanced colorectal cancer (In contrast, albumin was strongly associated with the magnitude of the systemic inflammatory response as evidenced by circulating concentrations of C-reactive protein. Furthermore, in a model combining albumin, C-reactive protein and PHR, there was no relationship between albumin and PHR when adjusted for the effect of C-reactive protein. Therefore, the results of the present study are consistent with the concept that the systemic inflammatory response is a major determinant of albumin concentrations in patients with advanced colorectal cancer.It may be that the presence of a chronic systemic inflammatory response, with its increased demand for specific amino acids for acute phase protein synthesis, promotes the degradation of available body protein including albumin (The results of the present study also showed that increased activity of the enzymes alkaline phosphatase, aspartate transaminase and alanine transaminase reflect increased tumour burden. In particular, raised alkaline phosphatase activity, a recognised prognostic factor, was significantly correlated with tumour volume and therefore its prognostic value would appear to be related to the size of the liver metastases. These increased enzyme activities are consistent with previous work in patients with colorectal liver metastastases (In summary, the results of the present study suggest that, in patients with advanced colorectal cancer, hypoalbuminaemia is secondary to the presence of a systemic inflammatory response rather than nutritional depletion by the tumour.


Metabolite ratios (phosphocreatine (PCr)/beta nucleoside triphosphate (beta NTP), PCr/Pi, beta NTP/Pi) calculated from the NMR spectra were compared with ratios obtained from acid extracts of tumours of similar size. Measurements of creatine and ADP were also made. Three of the tumours showed positive correlations between increasing tumour size and decreasing metabolite ratios measured both by NMR and in extracts, whereas the Walker carcinosarcoma showed no correlation between size and any parameters measured. Phosphorus metabolite ratios, measured in extracts of skin overlying the tumours, indicated a fall in high energy phosphate when there was histological evidence of skin invasion by the tumour. Surface coil 31P-NMR spectra of subcutaneously grown or induced tumours in the rat represent a slowly changing steady state as the tumour increases in size. We conclude that increasing numbers of hypoxic tumour cells, rather than large areas of necrotic tissue, contribute largely to the NMR spectrum.


These in vitro results prompted us to examine the effect of Epo-signal withdrawal on tumours in vivo. RT-PCR analysis demonstrated the expression of mRNAs for Epo and EpoR in the transplants of uterine and ovarian tumours in nude mice. Then we injected locally anti-Epo antibody or soluble form of EpoR into the transplants. At 12 h, 1, 7 or 14 days after the injection, all transplants were resected and examined macro- and microscopically. Tumour size was reduced in Epo signal-deprived transplants. Immunohistochemical examinations revealed destruction of Epo-responding malignant and capillary endothelial cells through apoptotic death. The degree of tumour regression correlated well with the dose and frequency of the injections. Control xenografts with saline injection or needle insertion showed well-developed tumour masses. This Epo response pathway will have profound implications for our understanding of the development and progression of malignant tumours and for the use of Epo-signal deprivation as an effective therapy. © 2001 Cancer Research Campaign


A method of comparing differences in growth rates of tumours in small groups of animals is described. A common slope can be fitted to the growth curves of a given tumour in a group of isogeneic animals. Differences between growth potentials can be demonstrated by comparing the common slope for a given tumour against that of another tumour (or the same tumour at a later stage of development).A highly significant difference is shown between the growth potential of an A-strain mammary carcinoma after 7 or after 28 days' growth in isogeneic animals. Since this increase in autonomy is reflected in the tumour's subsequent growth rate in secondary hosts, it is suggested that it involves adaptation of the tumour rather than progressive immunodepression of the primary host.


Tumour volume in EGCG- (25 mg kg−1)+tamoxifen (75 μg kg−1)-treated mice decreased by 71% as compared with vehicle control (P<0.05), whereas tumour weight was decreased by 80% compared with control (P<0.01). Epigallocatechin gallate treatment did not alter ER protein expression in MDA-MB-231 cells and thus was not a mechanism for the observed tumour suppression. However, western blotting of tumour extracts demonstrated that epidermal growth factor receptor , pEGFR (78% lower than control), mammalian target of rapamycin , and CYP1B1 (75% lower than control) were significantly lower after the combination treatment as compared with all other treatments. Nuclear factor-κB (NF-κB), b-Raf, p-MEK, S6K, 4EBP1, Akt, vascular EGFR-1 (VEGFR-1) and VEGF expressions were decreased in control but not in the individual treatments, whereas MEK, phospholipase D 1/2, TGFα, and ERK expressions were not changed after any treatment. The results demonstrate that tamoxifen at realistic doses (75 μg kg−1) can suppress the growth of ER-negative breast cancer when combined with EGCG. In addition, the dominant mechanism for tumour suppression is the concomitant decrease in tumour protein expressions of mTOR and the EGFR.We have previously shown that tamoxifen+epigallocatechin gallate (EGCG) is synergistically cytotoxic towards oestrogen receptor (ER)-negative breast cancer cells. To determine if this response would correlate with significant tumour suppression  Tamoxifen has been extensively used in the treatment of oestrogen receptor (ER)-positive breast cancer and was recently approved for chemoprevention in high-risk women . In ER-p3K–Akt pathway (3K–Akt pathway results in the activation of the transcription factor nuclear factor-κB (NF-κB) (constitutively active in ERα− breast cancers) and mammalian target of rapamycin (mTOR), an important regulator of protein synthesis . Acetic acid, disodium hydrogen orthophosphate anhydrous, EDTA, DMSO, potassium dihydrogen orthophosphate, and sodium citrate were purchased from BDH Laboratory supplies . Primary antibodies NF-κB, bRaf, TGFα, 4EBP1, p4EBP1, vascular endothelial growth factor receptor (VEGF), VEGFR-1, mTOR, PLD1, PLD2, S6K, pS6K, MEK, pMEK, ERK, pERK, ERα, and ERβ were purchased from Abcam . Foetal bovine serum was purchased from Life Technologies Ltd. . All other chemicals were the highest purity commercially available.Epigallocatechin gallate (99% purity) was purchased from the Cayman Chemical Company . MDA-MB-231 human breast cancer cells were purchased from ATCC . Matrigel and the primary antibodies Akt, pAkt, EGFR, pEGFR and CYP1B1 were purchased from BD Biosciences . Minimum essential medium, potassium chloride, NaHCO3. Cells were cultured in 75 cm2 flasks and incubated in 5% CO2/95% humidified air at 37°C.MDA-MB-231 and MCF-7 cells were maintained in minimum essential medium supplemented with 10% foetal bovine serum, 1% antibiotic/antimitotic solution, and 0.2% NaHCO6/50 μl matrigel), which were left to form palpable tumours. Tumour volume was measured weekly with electronic calipers (L W H). When the tumour volume reached 100 mm3, mice (10 per group) were randomly assigned to the various treatment groups. Mice were then treated daily with tamoxifen (50 or 75 μg kg−1), EGCG (25 mg kg−1), EGCG+tamoxifen, or a vehicle control for each drug (5 ml kg−1) for 10 weeks. Dosing solutions were prepared fresh each day. Tamoxifen was given through oral gavage, whereas EGCG was given intraperitoneally. On the account of extensive first-pass metabolism  was based on our previous study, which demonstrated that this dose was non-toxic and elicited a modest decrease in MDA-MB-231 tumour growth  were purchased from Hercus Taieri Resource Unit . They had free access to sterilised standard Reliance Rodent Diet  and water, and were housed four mice per cage and maintained at 21–24°C with a 12-h light/dark cycle in a specifically designed pathogen-free isolation facility and allowed to acclimatise for 1 week before experimentation. The University of Otago Animal Ethics Committee approved all procedures. Mice were inoculated into the right flank with ER-negative cells . Results are expressed as IU l−1.Food consumption and body weight were monitored daily throughout the treatment period. Mice were euthanised by COμM) or estradiol (100 nM). Cells were harvested using a cell scraper, washed, and incubated in RIPA buffer , incubated on ice for 15 min, and centrifuged at 13 000 r.p.m. for 10 min at 4°C. The supernatant was collected and the protein concentration was determined using the BCA assay.MDA-MB-231 and MCF-7 cells were plated (200 000 cells per well) in six-well plates and treated for 24 h with EGCG , fixed in acetone, and air-dried overnight. Sections were then washed twice in Tween 20–PBS, incubated with normal serum for 30 min at room temperature, and then incubated overnight with primary CYP1B1 or VEGFR-1 antibody. Slides were then rinsed and peroxidase blocked using hydrogen peroxide (3%) before incubation with the appropriate secondary antibody for 30 min at room temperature in a humidified chamber. The sections were then incubated with ExtrAvidin  (1 : 20) for 30 min at room temperature in a humidified chamber before development with 3,3′-diaminobenzidine tetrahydrochloride as the chromogen and counterstaining with Mayer's haematoxylin. Once slides were dehydrated, DPX mounting medium and coverslips were applied. The sections were analysed from tumours obtained from each mouse and a representative slide is shown in the results section.Tumours were embedded in OCT compound and then sectioned  were analysed using an one-way ANOVA coupled with a Student–Newman–Keuls post hoc test, in which P<0.05 was the minimum requirement for a statistically significant difference.For all parameters where the variances were significantly different between control and treated mice , the data were log-transformed before statistical analysis was performed. Tumour growth experiments were analysed using a repeated measures two-way ANOVA coupled with a Student–Newman–Keuls −1) was combined with tamoxifen (50 μg kg−1), the growth rate of the tumours in the combination treated mice lagged behind the other treatments, but an overall significant decrease in tumour growth was not observed . Tumour weight was also decreased significantly after combination treatment  (−1).When EGCG (25 mg kgobserved . A signieatments . After 1ctively) . Importaα expression in cell lines with a low ERα expression (μM) or estradiol (100 nM) for 24 h, and MCF-7 cells were treated with estradiol (100 nM) as a control. The results demonstrated that estradiol increased the protein expression of both ERα and ERβ in MCF-7 cells, whereas neither EGCG nor estradiol induced the protein expression of the ER in MDA-MB-231 cells (As EGCG has upregulated ER31 cells .P<0.001) (P<0.001)   in tumours from combination-treated mice were all significantly decreased in control, whereas p-Akt and NF-κB were also decreased in control and EGCG  . A similP<0.001) . Howevereatments . Given teatments . However(P<0.05) . Other dand EGCG .P<0.001)  . HoweverP<0.001) . PhosphoP<0.001) .P<0.05). To gain insight into the role of angiogenesis in the growing tumour, VEGF, an important regulator of angiogenesis  and EGCG  inhibits the growth of MDA-MB-231 xenografts and that this effect is superior to that elicited by either individual treatment. Another study has shown that the combination of green tea extract (2.5 g/l) and tamoxifen (20 mg pellet) is better than either drug alone at suppressing the growth of MCF-7 xenografts, and this correlated with increased levels of apoptosis and a suppression of angiogenesis in tumour tissue  was minimally effective at reducing tumour growth in a MDA-MB-231 xenograft model, but acetylating EGCG and thus turning it into a pro-drug enhanced its efficacy (−1) was unable to suppress the growth of MDA-MB-231 xenografts, but the addition of OUS-03012 (100 mg kg−1) showed significant tumour suppression (decreased 50% in control). This result supports the use of tamoxifen in combination therapies for ER-negative breast cancer. However, our combination with EGCG produced a greater level of tumour suppression at a much lower dose of tamoxifen (75 μg kg−1).Our results demonstrated that tamoxifen on its own was not effective at suppressing ER-negative tumour growth, whereas EGCG had a modest effect on tumour growth, but the combination of the two drugs showed marked tumour suppression. Other studies have also shown that EGCG or green tea extract has a modest inhibitory effect on ER-negative tumour growth  and significant reduction of EGFR and mTOR occurred after combination therapy, it is most likely that this dual suppression is a key mechanistic component of the observed tumour suppression. Furthermore, the decreased expression of mTOR is not likely to be regulated further upstream through PLD1/2, as levels of PLD1 and PLD2 were not altered by any drug treatments. However, the combined inhibition of EGFR, Akt, and mTOR after EGCG+tamoxifen treatment provides a powerful suppression of tumour growth.The mechanism responsible for the enhanced tumour suppression and apoptosis (Studies with EGCG in a variety of models have demonstrated that it decreases angiogenesis . Our resin vivo administration of EGCG. However, this protein could be important in ER-negative breast cancer, as the CYP1B1_1358_A>G polymorphism has been significantly associated with ER-negatvie tumour status (Another important effect elicited by the drug combination is the 78% decrease in the protein expression of CYP1B1. CYP1B1 has not been examined previously following the In summary, we have demonstrated that the combination of EGCG+tamoxifen significantly reduced ER-negative tumour growth and that this was driven primarily by an enhanced effect by the decreased protein expression of the EGFR, mTOR, and CYP1B1. Overall, our findings suggest that EGCG+tamoxifen as an antitumorigenic therapy in ER-negative breast cancer is promising and thus this combination warrants further investigation.


Prostaglandin (PG) E2 biosynthesis in Yoshida hepatoma (AH 130) was evaluated by radioimmunoassay. When hepatoma cells were incubated in vitro, the levels of PGE2 in the medium were similar to those found in hepatocytes for the first 2 h; this was followed by a rapid increase in PGE2 formation, and the 6h incubation levels were 4-fold higher than in hepatocytes. Addition of sodium arachidonate markedly and dose-dependently stimulated PGE2 synthesis; the increase was largely prevented by the addition of indomethacin (1 microM) or L 8027, a prostaglandin synthetase inhibitor. Experiments in vivo indicated that indomethacin treatment of tumour-bearing rats significantly reduced the tumour mass. When rats were injected with PGE2 after receiving the drug, the number of tumour cells was very similar to that of untreated animals. This, as well as the inhibition of tumour growth by acetylsalicylic acid, strongly suggests that the inhibition of PG biosynthesis by anti-inflammatory drugs and the inhibition of tumour proliferation may be closely associated events. It was also found that injections of indomethacin very significantly prolonged survival of hepatoma-bearing rats. Since PGE2 does not appear to affect the cyclic AMP levels of hepatoma cells, it is possible that hepatoma may use PGE2 to subvert the immune system. This could help to explain the effectiveness of anti-inflammatory drugs in the control of tumour growth.


An intravenously administered fluorescent carbocyanine dye, DiOC7(3), has been evaluated for use in conjunction with Hoechst 33342 as a marker of murine tumour vasculature. DiOC7(3) stains cells immediately adjacent to blood vessels and thus, like Hoechst 33342, outlines perfused tumour vasculature. The different fluorescence excitation and emission properties of DiOC7(3) and Hoechst 33342 permit discrimination of the stains in the same tissue section. Mice tolerate a DiOC7(3) dose of 1 mg kg-1 i.v. with no ill effects. The dye has a distribution half-life in blood of 180s and staining of perivascular tumour cells is sufficiently stable to allow visualisation of vasculature for up to 30 min after DiOC7(3) injection. However, DiOC7(3) causes a 75% reduction in tumour blood flow as measured by laser Doppler techniques. Consequently, the compound appears to be most suitable as a second vascular marker, administered at some time after Hoechst 33342, to detect temporal and spatial fluctuations in tumour perfusion.


We assessed factors which affect cisplatin concentrations in human surgical tumour specimens. Cisplatin 10 mg m-2 was given i.v. to 45 consenting patients undergoing surgical resection of neoplasms, and platinum was assayed in resected tumour and in deproteinated plasma by flameless atomic absorption spectrophotometry. By multiple stepwise regression analysis of normalised data, patient characteristics that emerged as being most closely associated (P < 0.05) with tumour platinum concentrations (after correcting for associations with other variables) were tumour 'source' [primary brain lymphomas, medulloblastomas and meningiomas ('type LMM') > 'others' > lung cancer > head/neck cancer > gliomas) or tumour 'type' , serum calcium and chloride (positive correlations) and bilirubin (negative). Tumour location  did not correlate with platinum concentrations. If values for a single outlier were omitted, high-grade gliomas had significantly higher platinum concentrations (P < 0.003) than low-grade gliomas. For intracranial tumours, the computerised tomographic scan feature that correlated most closely with platinum concentrations in multivariate analysis was the darkness of peritumoral oedema. Tumour source or type is a much more important correlate of human tumour cisplatin concentrations than is intracranial vs extracranial location. Serum calcium, chloride and bilirubin levels may affect tumour cisplatin uptake or retention. CT scan characteristics may help predict cisplatin concentrations in intracranial tumours.


A new method to test the sensitivity of tumour cells to chemotherapy is presented. Tumour cells were incubated in vitro on agar, and drug-induced cell cycle perturbation was monitored by flow cytometric DNA analysis. In the present study the method was applied to monitor the effect of adriamycin on an adriamycin-sensitive Ehrlich ascites tumour and two adriamycin-resistant tumours. Adriamycin caused a dose-related accumulation of tumour cells in the G2 + M phase in the sensitive tumour. Drug concentrations greater than or equal to 100-fold higher were required to induce similar changes in the resistant tumours. The dose level causing maximum accumulation in the G2 + M phase is suggested as a parameter for quantifying the sensitivity. The results indicate that the method can be extended to sensitivity testing of human tumours.


The mean tumour uptake of111In-labelled pegylated liposomes at 24 hours was 7.2 ± 6.6% ID/g. Liposome uptake for tumours < 0.1 g, 0.1–1.0 g and > 1.0 g was 15.1 ± 10.8, 5.9 ± 2.2 and 3.0 ± 1.3% ID/g, respectively. An inverse correlation between tumour weight and liposome uptake was observed by both Spearman’s rank correlation test  and Pearson’s correlation coefficient . For 18 tumours with macroscopic central necrosis, the ratio of uptake in the tumour rim relative to the necrotic tumour core was 11.2 ± 6.4. Measurement of tumour vascular volume for tumours of various sizes revealed an inverse correlation between tumour weight and tumour vascular volume , consistent with poor or heterogeneous vascularization of larger tumours. These data have important implications for the clinical application of pegylated liposome targeted strategies for solid cancers which are discussed in detail. © 2000 Cancer Research CampaignThe relationship between tumour size and uptake of


Growth of the Walker 256 carcinosarcoma implanted within various sites in Spraque-Dawley rats was investigated in animals receiving twice daily i.p. injections of the antiprotease aprotinin. Although administration of aprotinin partially attenuated the growth and lethality of i.p. tumour, no effect of aprotinin was found on intramuscular tumour development. Furthermore, we were unable to demonstrate unequivocal growth inhibition by aprotinin of lung tumour colonies from i.v. injection of tumour cells. Histological examination of intramuscular and pulmonary tumours revealed little evidence of host cellular immune response in either saline- or aprotinin-treated rats.


An experimental tumour system for the study of metastasis has been developed using a syngeneically transplanted murine squamous carcinoma of spontaneous origin. Implants of the tumour, which does not elicit a significant immune response, grew and metastasized regularly to regional lymph nodes and lungs, in a manner comparable with that of the more malignant types of human epithelioma. The system has been used to test the influece of pre-operative irradiation, regional lymph node excision, tumour biopsy and manipulation, on metastasis. Of these, only pre-operative irradiation with 2000 rad 24 h before tumour excision produced a significant differential effect--a lower incidence of metastasis. By contrast, local radiation therapy sufficient to cause complete tumour regression but insufficient to achieve long-term local cure was shown to result in accelerated metastasis. A highly significant inhibition of metastasis was observed with the drug ICRF 159, but histological features suggested that its anti-metastatic effect in this system did not depend on morphological changes which might prevent dissemination of tumour cells.


Direct comparison of skin-tumour induction by 15,16-dihydro-11-methylcyclopenta[a]phenanthren-17-one (I) and by benzo[a]pyrene on mouse skin, both by repeated application or by initiation with a single dose followed by promotion with croton oil, demonstrated that these two carcinogens have similar potency. After repeated application of (I) the mean latent period for skin-tumour induction was linearly related to the logarithm of the dose over a 10-fold dose range. Under these conditions, application of the aryl-hydrocarbon-hydroxylase inhibitor 7,8-benzoflavone together with (I) inhibited tumour induction by about 40%. By contrast, in the 2-stage experiment, little effect on tumour incidence or latent period was observed when this inhibitor was applied with the single initiating dose of (I). Co-administration of the epoxide-hydratase inhibitor 1,1,1-trichloropropene oxide caused enhancement by shortening the latent period. After s.c. injection of (I) into mice, a similar number of tumours was induced on skin remote from the site of injection by promotion with corton oil begun either one week or 6 months after initiation. Gastric instillation of (I) into female rats induced mammary adenocarcinomas.


The injection (s.c. or i.p.) of 10(6) live or lethally irradiated methylcholanthrene-induced fibrosarcoma cells into CBA/Ca mice one or 2 days before i.v. challenge with the same tumour inhibited the formation of artificial lung tumour metastases. In addition, it also frequently enhanced the cytostatic effect of peritoneal exudate cells on monolayers of the same tumour. The effects on lung tumour metastasis were not noted if X-irradiated tumour was injected i.v., or if s.c. administration was delayed until one day after i.v. challenge. Similar effects on tumour growth were also observed in C3Hf/Bu mice and (CBA/Ca x A/HeJ) F1 hybrids which were pretreated (s.c.) with tumour shortly before i.v. challenge with the same tumour. Further studies in CBA/Ca mice suggested that the protective effect was tumour-specific, for the growth of i.v. injected tumour was not significantly inhibited by pretreatement with a number of other MC-induced or spontaneous tumours from the same and different strains.


This factor might partly account for the discrepancy between human tumour growth rates  and the faster tumour growth rates observed in experimental animals, in which normally only single tumours are present.Spontaneous and transplanted tumour growth rates studied in the C3H mouse have shown that when only one spontaneous tumour was present in one strain then the distribution of growth rates closely resembled that for first generation isotransplants of another strain. It was also shown that the number of spontaneous tumours (in the range one to four tumours) present on the mouse affected the tumour growth rate,


Observations were made over a 4-year period on squamous cell carcinomas on the ear and other areas poorly covered by hair or wool, sheep pastured in the hot, dry environment of north-western Queensland. Overall incidence in the flock was higher than in flocks kept at greater latitudes. Increased incidence with advancing age was demonstrated, and ewes appeared to be more susceptible than wethers. Metastases were observed in 4 of 33 affected ewes (12%) submitted to detailed necropsy examination. Measurements of tumour growth in 4 ewes revealed an increase in size of about 3-5 mm per month. Ovine aural squamous cell carcinoma was considered to be a good model for studies on skin cancer in man.


In vitro cytotoxicity test showed that sheep anti-Ehrlich ascites tumour IgGl fraction was cytotoxic to 51Cr labelled tumour cells whereas IgG2 had no cytotoxic effect. Pretreatment of the tumour cells with the non-cytotoxic IgG2 fraction slightly inhibited the cytotoxic action of IgG1 in vitro.Hyperimmune heterologous serum produced in sheep against mouse Ehrlich ascites tumour cells was absorbed with normal mouse tissue and fractionated by DEAE column chromatography into IgG1 and IgG2 fractions. in vitro before injecting intraperitoneally into mice, both fractions protected the animals against tumour growth. Injection of IgG2 and IgGl fractions separately, one before and the other after the injection of EAT cells, resulted in partial protection only. The difference encountered between the in vitro and in vivo findings could be attributed to the host defence mechanisms involved in the in vivo test system.When EAT cells were coated with either IgG1 or IgG2 by preincubation


Nuclear polyploidization in the livers of CF-1 mice, exposed to dieldrin , was studied up to the median time of liver tumour development (ranging from 15 to 27 months) in the respective treatment groups. In untreated controls nuclear polyploidization is characterized by a linear increase of octaploid nuclei with age. Approximately 4 months before tumour development a reduction in the tetraploid to diploid ratio is observed. Dieldrin treatment was found to enhance nuclear polyploidization in the initial phases of treatment, as expressed by a dose-dependent increase in octaploid nuclei. In 'steady-state' situations all age dependent changes in the level of polyploidization found in controls were also found in dieldrin treated mice. However, these changes occurred at an increasingly earlier age with higher dieldrin treatment levels. The decrease in the tetraploid:diploid ratio always takes place a few months before tumour development. This change in the ploidy level may thus be related to the subsequent liver tumour formation. The liver tumours themselves appear to originate from a diploid stem line, and were found to increase their degree of polyploidization during growth, eventually developing aneuploid nuclei. A comparison of nuclear polyploidization and liver tumour formation in CF-1 mouse liver for the given dietary dieldrin concentrations showed that liver tumour formation was associated with a constant level of polyploidization. Since polyploidization is an age-dependent process, these findings suggest that liver tumour formation is imminent at a constant biological age and that dieldrin may advance the biological age of CF-1 mouse liver.


Malignant effusions and tumour tissue obtained at surgery provided material for a study of the prognostic value of the various inflammatory cells in the prognosis of human ovarian cancer. Ascitic fluids predominantly contained inflammatory cells; tumour cells, both singly and in clusters, were a minor component. Tumour cells were usually in excess in dispersed solid material. Some patients had significant proportions of lymphocytes and macrophages in their solid tumour, and these patients invariably responded to therapy. Sedimentation-velocity separation at unit gravity provided tow populations of inflammatory cells. One consisted of mononuclear cells similar in size to those in the patients blood: the other consisted of one or more large macrophage populations, distinct in morphology and enzymatic markers from both blood monocytes and each other. T lymphocytes were enriched in ascites fractions (78%) and in the tumour-derived mononuclear fraction (71%) compared to patient blood (60%). The T-cell subset characterized by ANAE reactivity was markedly depleted in the tumour-infiltrating fraction (17%) compared to patient blood (62%) or patient blood (51%). Esterase-positive monocyte-like cells were more frequent in the tumour-infiltrating fraction (17%) than ascites (7%) or blood (12%). B lymphocytes were infrequent in solid tumours and difficult to assess in ascites. Histiocyte-like macrophages were present in the higher-velocity tumour-cell containing fractions of both solid and ascitic material. The variation in infiltrating cells between patients and between tumour and ascites of the same individual was marked.


Extrapolation to humans from experimental radioimmunotherapy in nude mouse xenograft models is confounded by large relative tumour size and small volume of distribution in mice allowing tumour uptake of radiolabelled antibodies unattainable in patients. Our large animal model of human tumours in cyclosporin-immunosuppressed sheep demonstrated tumour uptake of targeted radiolabelled monoclonal antibodies comparable with uptakes reported in clinical trials. Sheep immunosuppression with daily intravenous cyclosporin augmented by oral ketoconazole maintained trough blood levels of cyclosporin within the range 1000-1500 ng ml(-1). Human tumour cells were transplanted orthotopically by inoculation of 10(7) cells: SKMEL melanoma subcutaneously; LS174T and HT29 colon carcinoma into bowel, peritoneum and liver; and JAM ovarian carcinoma into ovary and peritoneum. Tumour xenografts grew at all sites within 3 weeks of inoculation, preserving characteristic morphology without evidence of necrosis or host rejection. Lymphatic metastasis was demonstrated in regional nodes draining xenografts of melanoma and ovarian carcinoma. Colonic LS1 74T xenografts produced mucin and carcinoembryonic antigen (CEA). The anti-CEA IgG1 monoclonal antibody A5B7 was radiolabelled with iodine-131 and administered intravenously to sheep. Peak uptake at 5 days in orthotopic human tumour transplants in gut was 0.027% DI g(-1) (percentage of injected dose per gram) and 0.034% DI g(-1) in hepatic metastases with tumour to blood ratios of 2-2.5. Non-specific tumour uptake in melanoma was 0.003% DI g(-1). Uptake of radiolabelled monoclonal antibody in human tumours in our large animal model is comparable with that observed in patients and may be more realistic than nude mice xenografts for prediction of clinical efficacy of radioimmunotherapy.


The anti-tumour effect induced by a delayed hypersensitivity response (DHSR) unrelated to the tumour or by intra-tumoural inoculation of BCG was studied with 6 syngeneic mouse tumours. The growth of the tumours was followed i.p. or s.c. in suitably sensitized animals either in the presence or absence of the specific antigen required to elicit a DHSR. In a Winn-type assay the growth of tumour cells admixed with sensitized lymphocytes was also determined with and without the eliciting antigens. In addition, the effect of admixing different amounts of BCG with the tumour cells was studied on the growth of the tumours in vivo. The different tumours varied widely in their susceptibility to growth inhibition by a DHSR reaction and by BCG but their order of sensitivity was the same in all of the tests. Analysis of the effector population in the Winn test coupled with the inability to observe an anti-tumour action in mice with defective T-cell function showed that the effector mechanism involved allergized T-cells or more probably products released when these were confronted with the specific antigen. In vitro the relative susceptibility of the different tumour cells to killing by activated macrophages and by NK cells was quite different to that found for in vivo growth inhibition but the in vitro response to lymphotoxin of the different tumours paralleled that produced by inflammation in vivo.


The effects of local tumour radiation over the dose range 7.5-30 Gy on the growth and cell kinetics of rat hepatoma H-4-II-E have been investigated. A plot of growth delays against log surviving fraction was linear below a fraction of 0.03, but failed to extrapolate to the origin. Following a single dose of 15 Gy to the tumour, DNA-precursor incorporation, labelling and mitotic indices were depressed for 7 days. Tumour cellularity, measured as DNA/g tumour, was reduced and the rate of increase of total clonogenic cells slower than after complete tumour recovery. From Day 7 to Day 9 all indices of proliferation recovered to about control levels, clonogenic cell numbers increased more rapidly and tumour cellularity was restored. Repopulation of the tumour therefore appeared to take place mainly after Day 7. Incorporation of [3H]-TdR into tumour DNA reached twice the control values on Day 9. The rate of tumour growth accelerated after the initial decrease, and maximum tumour growth rate was also twice the control values on Day 13. Accelerated growth rates in irradiated tumours, above those of control tumours, occurred 10-16 days after treatment. The effectiveness of sequential therapy may therefore be improved if given during this period of of accelerated tumour growth.


Levels of carcinoembryonic antigen (CEA) and glucose phosphate isomerase (GPI) have been compared in the circulating blood of hamsters bearing intra-muscular grafts of GW-39 human colonic tumour. CEA in the sera of GW-39 tumour-bearing hamsters ranged from 2-6 to 8-4 ng/ml (mean = 4-5 +/- 1-7 ng/ml). GPI in the sera of normal hamsters ranged from 332 to 749 iu/1 (mean = 602 +/- 110 iu/1) while those with 14-week-old intra-muscular grafts of a hamster amelanotic melanoma, (A.Mel.3), or GW-39 human colonic carcinoma had a range of 664 to 1267 iu/1 (mean = 1024 +/- 220 iu/1) and 1430 to 4719 iu/1 (mean = 2065 +/- 601 iu/1) respectively. Thus, the ratio of enzyme activity in GW-39, A.Mel.3, and normal hamsters was 3-4:1-7:1, indicating a significant elevation (P less than 0-01) in animals bearing a human colon carcinoma or a hamster melanoma, with particularly high values obtained in hamsters with GW-39. Sequential determinations of CEA and GPI in a group of hamsters transplanted intra-muscularly with GW-39 tumours revealed that both markers increased proportionately with duration of tumour growth, suggesting that both serum CEA and GPI may be used as measures of tumour growth. The concentration of GPI in GW-39 human colonic carcinoma xenografts was also significantly higher than that measured in normal human colon, primary human colonic cancer, or normal hamster tissues. These results support the view that GPI, in addition to CEA, is a quantitatively increased marker in this tumour model, and is liberated into the circulation in proportion to the increase in tumour mass.


The distribution of iodine-131 (131I) labelled antibody to carcinoembryonic antigen (CEA) has been studied in 16 patients with colorectal cancer. Levels of tumour and normal tissue radioactivity were measured by serial gamma-camera imaging and counting of blood and urine. Maximum concentrations were found in tumour 8 h after administration and varied up to 9-fold in different patients. Higher levels were found on average in tumour than in any other tissue. Liver, lung and blood were the other tissues in which antibody was concentrated relative to the rest of the body. Antibody cleared from all these tissues over 1 week. Second antibody directed against the antitumour (first) antibody was given 24 h after first antibody in order to accelerate clearance from the blood. This increased the tumour to blood ratio but had little effect on other tissues. Cumulative radiation dose to tumour and normal tissue was estimated. In patients with the most efficient localisation the tumour to body ratio was 20:1 and tumour to blood ratio 5:1. This may be sufficient for effective therapy of cancer in patients selected for efficient antibody localisation. The data may be used to estimate the effect of different therapeutic strategies. For instance, in the time after second antibody administration the average tumour to blood ratio of radiation dose was 11:1, suggesting that two phase systems in which the therapeutic modality is given after a good tumour to normal tissue ratio is obtained may be effective for the majority of patients.


Tumour samples from 150 patients with squamous cell carcinoma of the oesophagus were investigated immunohistochemically with the monoclonal antibody MIB-1, which recognises proliferating cells. Using light microscopy, the number of MIB-1-positive tumour cells was counted in the areas with the highest proliferative activity. The MIB-1 index was determined as the proportion of MIB-1-positive and MIB-1-negative tumour cells. A considerable variation of the MIB-1 indices was found between the different tumours with a minimum of 6% and a maximum of 95% . The MIB-1 index correlated significantly with the mitotic activity in the tumour tissue  and with the proportion of apoptotic tumour cells . No significant correlation was found between the MIB-1 index and various other prognostic parameters including pT classification, pN classification, tumour grade, blood vessel invasion and lymphatic vessel invasion. In the univariate survival analysis no significant difference was found between tumours with low (< or = 33%) and high MIB-1 index (> 33%) 5-year survival rate: low MIB-1 index, 19.2%; high MIB-1 index, 22.2%). In a Cox proportional hazard regression analysis only the parameters lymphatic vessel invasion (P = 0.0001), pT classification (P = 0.0034) and pN classification (P = 0.0256), but not the MIB-1 index, could be verified as independent prognostic variables. In conclusion, evaluation of the MIB-1 index does not provide prognostic information for oesophageal cancer patients.


The carcinogen, methylcholanthrene was injected subcutaneously into mice and the steady development of fibrosarcomas was observed over approximately 200 days. These fibrosarcomas were strikingly infiltrated with FoxP3+ regulatory T cells implying that these cells impinge upon immune-mediated rejection of the tumour. This was confirmed by partial ablation of FoxP3+ regulatory T-cell activity, which resulted in a marked reduction in tumour incidence. The reduction of tumour incidence was ablated in mice that lacked interferon gamma. These data offer strong support for the concept of immune surveillance and indicate that this process is limited by the inhibitory effect of FoxP3+ regulatory T cells.Burnet proposed in the 1950's that the immune system is engaged in identifying and destroying abnormal cancerous cells. This process, termed immune surveillance, has been at the centre of intense debate for decades. Results using immunodeficient mice lend support to the immune surveillance hypothesis. We surmised that immune surveillance would be hampered by the inhibitory effect of naturally occurring FoxP3 In humans, the regulatory CD4+ T-cell pool is restricted to 1–2% of the CD4+ population that are described as CD25hi  is an established tumour induction model that has been used to examine the role of a series of cell types and signalling molecules that suppress tumour development. Thus, using MCA, we have examined how altered frequencies of Tregs impinge on the development of de novo tumours. Specifically, we determined whether (1) Tregs are present in MCA-induced tumours, (2) Tregs influence de novo tumour development, (3) IFNγ is required for control of tumour growth in Treg depleted mice and (4) depletion of Tregs promotes autoimmunity in MCA-treated mice. The implications of our findings are discussed in the context of tumour immune surveillance.CD4s CD25hi . An abseγ-deficient (IFNγ−/−) mice on a C57BL/6  background were sourced on site. IFNγ−/− mice were originally purchased from the Jackson Laboratory  and kindly donated by Professor N. Topley. Mice were housed in filter top cages and supplied with sterile food and bedding. Experiments were performed in compliance with UK Home Office regulations.Six- to twelve-week-old female wild-type (WT) and IFNμg MCA  suspended in 100 μl olive oil was injected subcutaneously (s.c.) into the hind leg of the mice. Mice were monitored weekly for tumour development.A total of 400 Mice were injected intraperitoneally (i.p.) with 0.5 mg of the CD25-specific monoclonal antibody (mAb), PC61 . Cells were stained with anti-CD4-fluorescein isothiocyanate (FITC), anti-CD4-PE Alexa 610, anti-CD25-biotin (7D4)/SA-PerCP-Cy5.5, anti-TCRαβ Allophycocyanin (APC), anti-FcγIII/II receptor  and anti-FOXP3-R-Phycoerythrin (PE) using the ebioscience staining kit  mAbs.Single-cell suspensions were prepared by filtering mashed tissues through 70 + T cells were purified from single-cell suspensions of splenocytes by magnetic associated cell sorting (MACS) . CD4+CD25+ cells were separated from CD4+CD25− cells by MACS. CD4+CD25− cells (2 × 104) were stimulated with 1 μm anti-CD3 mAb  and 1 × 105 mitomycin  treated CD4− splenocytes and incubated with titrated numbers of CD4+CD25+ cells.CD4μm) were dried and fixed with cold acetone for 15 min on ice. Sections were permeabilised with 0.2% Tween 20, 1% BSA for 30 min before a 1-h incubation with rabbit anti-mouse foxp3 polyclonal antibodies (Ab)  IgM autoantibody according to the manufacturers’ protocol .t-test. Comparisons of tumour induction between experimental groups were made using a log-ranked test.The extent of Treg infiltration in different organs was compared using a paired μg) of MCA. We first sought to determine whether Treg infiltrate MCA-induced tumours. Methylcholanthrene-induced tumours were removed from mice and examined histologically for Tregs by staining sections with Abs specific for FOXP3 and CD4. As shown on +FOXP3+ Tregs, which were in close proximity to CD4+FOXP3− conventional T cells , TILs were stained with CD4-, CD25- and FOXP3-specific Abs and analysed by flow cytometry. Supporting the histological observations, approximately half of the CD4ed FOXP3 . Approxied FOXP3 , a findiess CD25 . A signied NTDLN . CollectThe markedly high infiltrate of Tregs within the fibrosarcomas led us to explore whether depletion of the Tregs would protect against the development of MCA-induced tumours.+FOXP3+ Tregs in spleens of treated mice  . This failure to enhance tumour rejection may however be attributable to codepletion of activated nonregulatory T cells.Mice treated as described above with either PC61 or control mAbs, were injected 1 day later with MCA mixed in olive oil. Tumour growth was monitored over 200 days. P=0.001) . Next, wγ plays an important role in controlling development of MCA-induced tumours . These data imply that expansion and/or migration of Tregs in response to a developing tumour is impaired in the absence of IFNγ.IFN compare . To gainrol mAbs . Interes WT mice . SimilarAs a reduction in Treg numbers has been associated with the development of autoimmunity, circulating autoantibodies were analysed in mice that remained tumour free following Treg depletion. Mice treated with PC61 had a significantly elevated concentration of circulating dsDNA-specific IgM autoantibodies compared to mice that were not injected with PC61; however, there was no difference in dsDNA-specific autoantibody levels in mice treated with PC61 and that remained tumour free compared to tumour-bearing mice that received PC61 .in vivo. In this study, we used MCA, a carcinogen that has been widely used to identify a role of a series of immune cell types and signalling molecules that suppress tumour development. The study revealed several interesting findings. We first found a significant enrichment of CD4+FOXP3+ Tregs in tumours developing in vivo, where approximately half of total CD4+ TIL were FOXP3+. Moreover, injection of mice with the CD25-specific mAb, PC61, resulted in a transient depletion of a proportion of the Tregs before MCA injection and a significantly reduced tumour incidence. We believe that interesting parallels can be drawn between this work and a study performed 20 years previously by + T cells, capable of suppressing the activity of autoreactive T cells, begins only on day 3 after birth in mice (The aim of the work described here was to directly investigate whether Tregs impinge on immune surveillance of developing tumours The key question arising from our data relates to the type of effector cells promoting tumour rejection after depletion of Tregs. As Tregs have been shown to suppress both innate and adaptive immune responses to tumours (reviewed by γ in controlling the development of MCA-induced tumours (γ can control tumour growth directly through proapoptotic, antiangiogenic and antiproliferative effects and indirectly, by facilitating induction of antitumour innate and adaptive immune responses (reviewed by γ. This may be because immune responses uncovered by Treg depletion mediate tumour rejection through production of IFNγ and/or that immune responses uncovered by Treg depletion cannot compensate for the lack of IFNγ, which is critical for tumour control even in the absence of Tregs. Interestingly and in contrast to our observations in wild-type mice, tumours appeared slightly yet significantly earlier in IFNγ−/− mice receiving CD25-specific mAbs compared to those that received control mAbs. This finding suggests a fundamental difference in the response of wild-type and IFNγ−/− mice to MCA injection. It is possible that CD25-specific mAbs deplete important tumour-induced effector cells in IFNγ−/− mice. Alternatively, the impact of Treg activity on tumour development may differ between IFNγ−/− and wild-type mice. Tregs have been shown in some experimental models to suppress tumour progression by inhibiting inflammatory responses that would otherwise promote tumour development (γ−/− mice, thus the overall influence of Tregs would be to suppress tumour progression through limiting these responses. We did however observe that IFNγ−/− mice, when challenged with MCA, demonstrate a reduction of the percentage of CD4+FOXP3+ Tregs within tumours and TDLN. As mentioned earlier, this may reflect a role for IFNγ in expansion/migration of Treg cells or an overall increase in the expansion of effector T cells in the IFNγ−/− animals. Interestingly, the function of Tregs has previously been shown to be impaired in these mice (γ is important for the conversion of CD4+CD25−FOXP3− cells to CD4+CD25+FOXP3+ Tregs, thus it is possible that those CD4+FOXP3+ Tregs in MCA-induced tumours are derived from CD4+CD25−FOXP3− cells by a process, which is IFNγ dependent.Previous studies have highlighted a critical role for IFNAn accumulation of data obtained in studies of patients with cancer does support the concept that Treg depletion will have a beneficial effect in cancer immunotherapy. Overall, the results of this study support this premise by revealing a role for Tregs in suppressing effective immune surveillance of carcinogen-induced tumours in intact animals. A more cautionary note, implied by the findings of this study is that the nature of the ongoing immune response to the tumour may alter the outcome of Treg depletion, in some cases favouring tumour progression rather than tumour control. Further studies are clearly required to clarify this issue.


The ability of human tumour cells to form colonies in soft agar is enhanced by the presence of autologous phagocytic/adherent cells. We investigated the effect of irradiation on the ability of the adherent cells to support human tumour colony formation. Relatively low doses of irradiation significantly increased the growth enhancing ability of adherent cells in 17/19 cases. The possibility that the enhancement was mediated by inactivation of radiosensitive contaminating lymphocytes was explored. Depletion of T lymphocytes from unirradiated adherent cells by a monoclonal antibody and complement resulted in little overall change in tumour colony growth. However, elimination of only the suppressor subset (OKT8+) of T lymphocytes resulted in increased colony growth relative to control values obtained with unirradiated adherent cells. In contrast, depletion of T lymphocytes from irradiated adherent cells by a pan T monoclonal antibody and complement decreased colony formation. Thus, the ability of irradiated macrophages to enhance tumour colony growth appeared to be mediated by a T lymphocyte. The effect of irradiation on isolated populations of macrophages and T lymphocytes was also examined. The enhanced ability of irradiated adherent cells to support tumor colony growth appeared to have been due to treatment of T lymphocytes alone. The results indicate that both adherent macrophages and lymphocytes may influence the growth of clonogenic human tumour cells.


The kinetics of development and subsequent growth of a C3H mouse mammary tumour after implantation of 10(6) cells was quantified by observation and statistical analysis of latent period and growth rate for different categories of tumour. These comprised control tumours, tumours recurring after large single doses of X-rays alone and in combination with misonidazole, tumours developing from cells implanted both outside and within the sites of tumours previously cured by irradiation, tumours developing from cells implanted in heavily irradiated skin, and tumours developing from cells taken from tumours recurring after irradiation and re-implanted in untreated skin. The kinetics of development and growth of tumours in host animals previously treated by i.p. injection of killed tumour cells was also quantified. The results confirm that tumour development and growth is significantly perturbed by irradiation of host tissues both before and after tumour transplantation, and that this perturbation involves an extended latent period, a slower average rate of growth and a less uniform pattern of growth. These effects result from localized radiation damage to host tissues, are not attributable to residual damage to irradiated tumour cells, and are not markedly dose-dependent within the dose range 25--80 Gy. These results are consistent with the complete sterilization of host endothelial cells by doses of 25 or more. In marked contrast to the growth-slowing effect of irradiation, the treatment of host animals by previous injection of radiation-killed tumour cells led to a reduced latent period and a faster average rate of growth.


In patient tumour samples the activity in vitro of Taxol corresponded fairly well to the known clinical activity and Taxol showed low cross-resistance to standard cytotoxic drugs. However, the Taxol solvent Cremophor EL--ethanol was considerably active alone, whereas paclitaxel formulated in ethanol was less active. Taxol thus seems to contain two components active against patient tumour cells in vitro.