The combination of ionizing radiation with the pro-apoptotic TRAIL receptor antibody lexatumumab has been shown to exert considerable synergistic apoptotic effects in vitro and in short term growth delay assays. To clarify the relevance of these effects on local tumour control long-term experiments using a colorectal xenograft model were conducted.Colo205-xenograft bearing NMRI (nu/nu) nude mice were treated with fractionated irradiation  and lexatumumab . The tumour bearing hind limbs were irradiated with graded single top up doses at d8 under normoxic (ambient) and acute hypoxic (clamped) conditions. Experimental animals were observed for 270 days. Growth delay and local tumour control were end points of the study. Statistical analysis of the experiments included evaluation of tumour regrowth and local tumour control.Combined treatment with irradiation and lexatumumab led to a pronounced tumour regrowth-delay when compared to irradiation alone. The here presented long-term experiments revealed a highly significant rise of local tumour control for normoxic (ambient) (p = 0. 000006) and hypoxic treatment (p = 0. 000030).Our data show that a combination of the pro-apoptotic antibody lexatumumab with irradiation reduces tumour regrowth and leads to a highly increased local tumour control in a nude mouse model. This substantial effect was observed under ambient and more pronounced under hypoxic conditions. TNF-related apoptosis inducing ligand) receptor 2 (TRAIL-R2) induced apoptosis. Although TRAIL-R2 stimulation alone is highly effective in a wide range of cancer cell lines, efficacy can be increased by combination with other gyrostatic drugs (for review see [Lexatumumab is a fully human agonistic antibody with a distinct tumour cell specifity via activation of TRAIL (view see ). We havview see .Since discovery of TRAIL and its receptors in 1997 a panel of agonistic antibodies for TRAIL-receptors R1 and R2 have been developed and tested in clinical phase I and II trials -18. HoweCombining mapatumumab or lexatumumab with irradiation, we have demonstrated that this combination exerts strong additive and synergistic effects on apoptosis induction in vitro and in short-term growth delay experiments. HoweverTherefore, we decided to perform long-term experiments in a nude mouse xenograft model. As radiation sensitivity becomes affected by limiting intratumoural hypoxia we run experiments under both ambient and hypoxic conditions to mimic realistic tumour conditions.Taken together, our experimental series was designed to confirm the striking principle that radiation mediated TRAIL sensitization effectively increases long-term local tumour control.Immunodeficient NMRI-(nu/nu)-nude mice were purchased from a specific pathogen free colony at the University of Essen (Germany) at the age of 4-6 weeks. Animals were kept in an individually ventilated cage rack system  and fed with sterile high calorie laboratory food . Drank water was supplemented by chlorotetracycline and potassium sorbate acidified to a pH of 3.0 with hydrochloric acid.The Colo205 tumour cell line  was acquired from ATCC . In NMRI-(nu/nu)-nude mice Colo205 cells form solid, roundly shaped tumours without indication for metastasis.2)/2, where a and b are the long axis and the short axis, respectively. Scoring of tumour sizes took place three times per week before start of treatment. Body weight was monitored once a week.Tumour lumps of about 2 mm diameter from a source tumour were implanted subcutaneously into the right hind limb of 6-10 week old animals. Approximately 2-3 weeks after transplantation tumour growth was measurable. Tumour size was quantified with calipers in two perpendicular diameters. The tumour volume (V) was calculated as V =  irradiation. 3 Gy single dose was chosen for fractionated irradiation based on previous experiments . Fractionated irradiation of tumours was applied in inhalation (Isoflurane) narcosis. Top up irradiation under ambient conditions or under clamped hypoxia was performed with i.p. narcosis , as recommended by the university veterinarian department. For animals, whose tumours were clamped irradiation was performed 10 minutes after applying a narrow lace to the right hind limb just at the proximal end of the tumour to make the hypoxic radiation conditions as consistent as possible. Experiments were performed in one run with 252 animals.Tumour volumes were scored twice a week, no blinding took place. Follow up was discontinued after 270 days or in case of intercurrent death or if tumours had grown to eight-times the initial tumour volume at the start of treatment. Growth delay and local tumour control were endpoints of the study. All animal experiments were accomplished in accordance with the guidelines of the local authorities  and the German animal welfare regulations.Statistical analysis was performed as described before. In shorTreatment with lexatumumab failed to induce any immune reactions of the irradiated skin. No evidence of acute toxicity was observed. Follow up revealed no significant differences in frequency of intercurrent deaths after irradiation alone or combined treatment with lexatumumab (5.6% vs. 4.6%).Figure However, combination of very low doses of irradiation with lexatumumab led to an unexpected high local tumour rate, already. Tumour regrowth after combined treatment was observed in less than 50% of the animals. Figure Figure Our data prove that the combination of the proapoptotic human antibody lexatumumab with ionizing radiation has an obvious influence on local tumour control in a long-term xenograft model. The effect is evident after irradiation with low doses, already.It is important to note that these experiments with an agonistic antibody against TRAIL receptor DR5 corroborate our recently published data on a high efficacy of a combined treatment with another proapoptotic antibody  and irradiation. Both models are in line with in vitro data from our and other labs demonstrating that irradiation acts as a TRAIL sensitizer and not obversely,29,30.This principle diverges from other combined approaches where classical chemotherapeutic or other molecular targeted agents act as radiosensitizer. E.g. the synergizing efficacy of cisplatin is based on increased oxygenation of hypoxic cells and an influence in DNA-repair and cell cycle regulation -33. CetuIn contrast to former reports the mitochondrial pathway has a strong impact in TRAIL induced apoptosis. Depending on the cell system applied mitochondrial amplification loops account for its high efficacy,37. In cThe role of radiation induced TRAIL receptor upregulation has been discussed extensively. However, we and others found an only weak or lacking correlation between upregulation and synergism ,41,42. AIt is important to note, that this synergistic principle works under ambient and hypoxic conditions as well. Weinmann et al. demonstrated an undiminished efficacy of TRAIL alone under hypoxia in a lymphoma cell model. TakahasThe strong request on the development of personalized targeted therapies has amazingly changed the general approach to cancer treatment. In contrast to cytostatic drugs being prescribed on base of classical features as TNM classification and histology, targeted drugs require an accurate identification of patient collectives who benefit from a given treatment. Therefore, a specific subset of marker molecules should be identified for each targeted drug -48.The here presented data provide evidence that the combination of apoptosis inducing antibodies with irradiation strongly increases long-term tumour control. Since murine long-term control experiments are the only currently accepted functional approach to simulate the efficacy of radiation based treatments the given data are an optimal scientific base for subsequent clinical trials.The authors declare that they have no competing interests.PM conceived and drafted the manuscript. DJ and SS carried out the animal experiments to the same portion. WB performed the statistical analysis. MN participated in the statistical analysis and in the drafting of the manuscript. CB contributed to interpretation of the data and critically reviewed the article. All authors read and approved the final manuscript.


In order to provide a system suitable for our work, the model was changed by stepwise transitions to tumour specific immunoglobulin made from xenoantiserum absorbed in vitro, since such a procedure is also applicable to human patients. The time lapse used between challenge and treatment in the allo-system was generally ± 2 h but in the xeno-system could be extended to + 18 h. The xenoantisera could not be absorbed in vivo but required 3 to 5 × 103 spleens per 100 ml serum to absorb in vitro to render them tumour specific. The protective antibody was in the IgG (not IgM) fraction of serum. Maximal tumour specific antibody (measured by in vivo protection) appeared after the third injection of rabbits for one lymphoma, but after the fifth for another. The sera were not cross-reactive among 3 lymphomata tested, of which 2 were of the same H-2 genotype.Protection tests using passively administered antibody have been carried out using 2 mouse lymphomata. The classic model (“Gorer System”) used alloantiserum which was absorbed


The positive effect of castration in prostatic cancer patients is considered to be related to the induction of apoptosis in androgen-dependent tumour cells. However, castration apparently does not induce apoptosis in the highly differentiated, androgen-sensitive Dunning R3327PAP rat prostatic adenocarcinoma. To elucidate potential mechanisms of apoptotic induction in this tumour model, rats with subcutaneously implanted tumours were treated with vehicle (I), castration+vehicle (C) or castration + 50 micrograms of oestradiol benzoate per day s.c. (C + E2). The effects on tumours were examined by morphometry, in situ end labelling (ISEL) of apoptotic cells and immunohistochemically with monoclonal antibodies to proliferating cell nuclear antigen (PCNA) at different time points up to 168 h after castration. Castration inhibited tumour growth and decreased the epithelial cell apoptotic rate (from 12 h) and epithelial cell proliferation rate (from 72 h) compared with that in the I group. Tumour volume, volume densities of epithelium and stroma and stroma cell proliferation rate remained constant in the C group during the study period. C + E2 treatment resulted in increases in cell proliferation in the stroma (from 12 h) and in the volume density of stroma (from 24 h) compared with that in the C and I groups. The number of apoptotic epithelial cells was increased (from 24 h), and this was followed by decreases in the volume density of epithelium (from 24 h), the epithelial cell proliferation rate (from 72 h) and the total tumour volume (from 72 h). We conclude that in the Dunning R3327PAP tumour model C + E2 treatment is more effective than castration alone. C+E2 treatment, in contrast to C, is able to induce tumour cell death and to decrease total tumour volume. The mechanism behind this effect is unknown, but it could be related to stimulatory effects of E2 in the tumour stroma.


The light source used was an infrared-A irradiator, which, by use of appropriate filters, delivered the different ranges of wavelengths required. Following treatment, tumour volume was monitored. The greatest tumour growth inhibition was seen with Bchl-ser-PDT+HT, and subsequent experiments identified the pathophysiological basis for this effect. Red blood cell flux in tumour microvessels declined rapidly upon Bchl-ser-PDT+HT, reaching approximately 10% of initial values by the end of treatment. Similarly, tumour oxygenation worsened, reaching almost anoxic levels by the end of the treatment period. Assessment of metabolic parameters showed a pronounced increase in lactate levels and a decrease in ATP concentrations after combined treatment. The results presented suggest that vascular collapse and flow stasis resulting in a deterioration of tumour oxygenation and a switch from oxidative to glycolytic glucose turnover are key elements in the tumour eradication seen with this novel approach in which an antivascular PDT and HT are combined and simultaneously applied.The effects of combined and simultaneously applied localised 43°C hyperthermia (HT) and an antivascular bacteriochlorophyll-serine-based photodynamic therapy (Bchl-ser-PDT) on tumour growth and several microenvironmental parameters were examined. Rats bearing DS-sarcomas were allocated to treatment groups: (i) sham-treatment (control), (ii) Bchl-ser-PDT (20 mg kg In onco2 delivery  for enhanced antitumour therapy. Clinically, HT alone is not an appropriate option for the curative treatment of human tumours. However, a number of clinical studies have indicated a clear benefit when HT was combined with other therapy modalities, in particular radiotherapy (4 cells μl−1) into the hind foot dorsum of male Sprague–Dawley rats . Approx. 1 week after implantation, when a tumour volume of 0.7–1.0 ml had been reached, animals were anaesthetised with sodium pentobarbital . Throughout all experiments, animals lay supine on a thermostatically controlled heating pad and the rectal temperature maintained at 37.5–38.5°C. Animals breathed room air spontaneously. In the case of the acute experiments , polyethylene catheters were placed into the thoracic aorta via the left common carotid artery and into the right external jugular vein . Continuous monitoring of the mean arterial blood pressure (MABP) was possible via attachment of the arterial catheter to a pressure transducer . After the surgical procedures, animals were allowed to stabilise and treatment commenced once constant baseline readings for laser Doppler flux or oxygenation were obtained for at least 20 min.Solid tumours grew subcutaneously following injection of DS-sarcoma cells  and then diluted in 1 ml phosphate-buffered saline immediately prior to administration via a tail vein so that animals received a dose of 20 mg kg−1 body weight. In the case of combined Bchl-ser-PDT+HT, the photosensitizer was injected 10 min following commencement of HT. Correspondingly, Bchl-ser-PDT involved photosensitizer injection 10 min after commencement of illumination. Control animals received an equivalent volume of ethanol (400 μl kg−1) in 1 ml phosphate-buffered saline.The photosensitiser used in this study was Bchl-ser. This water-soluble derivative of the natural pigment bacteriochlorophyll-−1 i.v.) and ‘non-thermal’ illumination . Group III (HT): localised HT at 43°C . Group IV (Bchl-ser-PDT+HT): animals treated with Bchl-ser (20 mg kg−1 i.v.) and localised tumour HT for 60 min at 43°C .Group I (control): sham treatment. Group II (Bchl-ser-PDT): animals received Bchl-ser  was used to heat tumours (0.4°C min−1) to a set temperature of 43°C. Following the 20 min heat-up phase, the tumour temperature was maintained at 43°C for 60 min by regulation of the source which was switched on and off intermittently by the feedback control system, resulting in an average total energy dose of 1596 J cm−2 over the complete 80 min treatment period, as measured in pilot studies. These pilot studies were also used to obtain information concerning the pattern of irradiation and showed that, on average, the tumours used were irradiated for 1.3 s followed by a non-illumination period of 2 s over the 80 min treatment period. This pattern was subsequently used for the Bchl-ser-PDT treatment (Group II) so that the light delivered in the range from 665–800 nm was equal for all groups in which tumours were illuminated. The fluence rate for this wavelength range was 125 mW cm−2 and the total energy dose was 263 J cm−2. However, since the photosensitizer was administered 10 min after commencement of illumination (in order to correspond to the Bchl-ser-PDT+HT group), the ‘effective’ total energy dose  was 230 J cm−2. A radiometer/photometer  with a calibration traceable to the National Physical Laboratory (USA) was used for light dosimetry purposes. The irradiation was applied solely to the tumour, the remainder of the animal being shielded by aluminium foil.The radiation delivery system used has been described in detail previously . The eneIt should be noted that the light doses administered in the Bchl-ser-PDT group were not selected on the basis of conventionally used light doses in PDT, but rather arose from the properties of the light source used and the energy doses necessary to induce 43°C HT in those groups where tissue heating was induced.d) of the tumours were measured on a daily basis so that tumour volume (V) could be determined using the ellipsoid approximation V=π/6 × d1 × d2 × d3. The end point of the tumour response study was a tumour volume of 3.5 ml, rather than survival. This target value was selected to ensure that the tumour burden at the end of the study never exceeded 1% of body weight. Animals whose tumours did not reach the target volume were monitored for 90 days.The three orthogonal diameters (Determination of the red blood cell (RBC) flux in tumour microvessels was performed using a laser Doppler flowmeter . This method relies on the Doppler shift, assessing the frequency change undergone by light when reflected by moving objects, such as RBCs, and is a valid technique for measurement of microcirculatory function in small tissue areas , which was inserted into the tumour for continuous monitoring. Calibration of the pO2 electrode was carried out using room air in a constant temperature chamber, taking into account the prevalent barometric pressure. Data were subsequently expressed relative to the value obtained immediately prior to commencement of treatment.Polarographic assessment of tumour oxygenation was carried out using a flexible O2 immediately after completion of treatment. Two-thirds of the tumour mass was then ground to a fine powder and freeze-dried. Glucose and lactate concentrations were then determined using standard enzymatic assay kits , and global ATP, ADP and AMP concentrations were measured with reversed-phase HPLC, as described previously  were prepared from the remainder of the tumour. These were heat-inactivated at 80°C for 5 min before storage at −80°C. Microregional distributions of lactate and ATP concentrations were subsequently assessed using single-photon imaging and bioluminescence techniques . Bchl-ser-PDT alone also effectively inhibited tumour growth with a probability of 36% on day 90 (P<0.001). The greatest inhibition of tumour growth was seen upon Bchl-ser-PDT+HT, with a probability of tumours not reaching the target volume of 78%, 90 days after treatment (P<0.001). Of the tumours which did not reach the target volume within the observation period, the majority showed no evidence of tumour presence on macroscopic examination. Only one tumour treated with Bchl-ser-PDT+HT showed slow tumour recurrence from day 30 after treatment onwards, which still had not reached the target volume by day 90. The differences between the Bchl-ser-PDT+HT and Bchl-ser-PDT groups, while not being significant upon log-rank analysis, do suggest a trend towards a greater antitumour effect with the combined therapy form (P=0.083). Considering this, the subsequent experiments were carried out to elucidate the impact of the combined therapy on tumour perfusion, oxygenation and metabolic microenvironment, in comparison to the effects of HT alone, since such effects may play a key role in the observed inhibition of tumour growth.All animals recovered rapidly following treatment, with no phototoxic effects on normal skin being recorded. Assessment of tumour volume for up to 90 days provided the basis for a Kaplan–Meier analysis, showing the probability of the tumour volume being less than 3.5 ml during the observation period  in tumour microvessels and tumour oxygenation over the whole observation period are shown in Figure 3M were seen. When the lactate distribution images were compared, HT appeared to induce a slight increase in lactate levels. Upon Bchl-ser-PDT+HT treatment, distinct and extensive increases in lactate concentrations were observed, with numerous regions showing concentrations >40 mM.Examples of colour-coded images of microregional ATP and lactate distributions in tumour sections obtained using single-photon imaging and bioluminescence techniques are shown in Figure 4P<0.01), where the concentration of lactate almost doubled upon treatment. ATP levels were reduced upon treatment, with Bchl-ser-PDT+HT causing the most pronounced reductions (P<0.05). Global glucose levels were also assessed, although no significant changes were evident.The treatment-induced changes in lactate and ATP concentrations seen using single-photon imaging and bioluminescence techniques were confirmed using methods assessing global metabolite concentrations  of tumours not reaching the target volume. Of the tumours observed up to day 90, the majority showed no evidence of tumour presence, thus underlining the therapeutic potential of PDT combined with HT.in vitro and in vivo studies have suggested a possible role for HT in combination with conventional PDT   is a modality in which a number of treatment parameters related to the photosensitiser and light  used need to be selected, studies have also been carried out in which magnetic resonance spectroscopy (MRS) techniques have been used to try to predict PDT outcome at the time of treatment. In one such study (The combination of an antivascular PDT treatment with localised HT, as applied in this study, is based on a number of considerations. Firstly, Bchl-ser is injected i.v. 10 min after commencement of HT, at a time when the tumour is still in the heating-up phase and has a temperature of approximately 40°C. At this point in time a mean increase in tumour RBF flux of ≈10% was observed, which could therefore be exploited to enhance the delivery of Bchl-ser to the tumour tissue. Secondly, the photodynamic process is known to be inherently temperature dependent , and thuependent  and thusIn conclusion, an effective tumour therapy in which Bchl-ser-PDT and HT have been combined and simultaneously applied has been described, and the results presented suggest that vascular collapse and flow stasis resulting in a deterioration of tumour oxygenation and a switch from oxidative to glycolytic glucose turnover are key elements in the tumour eradication seen with this novel approach.


Purpose. Post-operative radiotherapy (RT) is routinely applied in the treatment of several human tumours. The aim ofthe present study was to investigate the value of post-operative RT in a rat model.            Methods. Experiments were performed using the rhabdomyosarcoma R1H of the WAG/Rij rat. Animals were randomizedto different treatment schedules: surgery, RT or a combination of both. Tumours were excised at different sizes (0.1–4.5 g)aiming for complete macroscopic resection. RT (60 Gy in 30 daily fractions over 6 weeks) was applied either primarily or tothe former turnout site from the third post-operative day. Tumour growth delay, time to recurrence and local tumour controlwere used as endpoints.            Results. Pre-operative tumour size determined the time and rate of recurrence. The larger the tumour,the shorter the time to relapse and the higher the recurrence rate. The 50% local control rate (LCR50) for surgery was found in tumourswith a mass of 0.8 g. For post-operative RT a LCR50 was achieved for tumours with a mass of 1.1 g. For larger turnouts(> 1.1 g), however, the rate and time course of relapse were similar for both the group receiving RT alone and the groupreceiving post-operative RT.            Discussion. In this model the tumour mass at excision governs the prognosis. Relatively small R1H turnouts may recurdespite complete macroscopical resection. With regard to the LCR, the outcome for larger tumours is improved withpost-operative RT (60 Gy/6 weeks) than compared with surgery alone. The factor is 1.3. Within a certain range of tumoursizes, combined treatment (surgery + RT) can improve the outcome considerably.


Furthermore, tumour hypoxia is thought to play a major role in the activation of macrophages and the regulation of angiogenesis. In this paper, we demonstrate a strong correlation between hypoxia and the recruitment of immune cells binding to IgG in 8 experimental tumours. We provide evidence that IgG binding immune cells in 3 tumour lines are predominately composed of macrophages. Reduced oxygenation may act as a stimulus for recruitment of immune cells to the tumour mass, and the detection of either IgG-positive host cells or macrophages may offer an alternative method for monitoring tumour hypoxia. © 2001 Cancer Research Campaign


Characteristics of the tumour metabolic profile play a role in both the tumour-host interaction and in resistance to treatment. Because carbogen (95% oxygen/5% carbon dioxide) breathing can both increase sensitivity to radiation and improve chemotherapeutic efficacy, we have studied its effects on the metabolic characteristics of Morris hepatoma 9618a. Host carbogen breathing increased both arterial blood pCO2 and pO2, but decreased blood pH. A fourfold increase in tumour pO2  and a twofold increase in image intensity [measured by gradient recalled echo magnetic resonance (MR) imaging sensitive to changes in oxy/deoxyhaemoglobin] were observed. No changes were seen in blood flow measured by laser Doppler flowmetry. Tumour intracellular pH remained neutral, whereas extracellular pH decreased significantly (P < 0.01). Nucleoside triphosphate/inorganic phosphate (NTP/Pi), tissue and plasma glucose increased twofold and lactate decreased in both intra- and extracellular compartments, suggesting a change to a more oxidative metabolism. The improvement in energy status of the tumour was reflected in changes in tissue ions, including Na+, through ionic equilibria. The findings suggest that the metabolic profile of hepatoma 9618a is defined partly by intrinsic tumour properties caused by transformation and partly by tissue hypoxia, but that it can respond to environmental changes induced by carbogen with implications for improvements in therapeutic efficacy.


In order to quantitate the effects of tumour subpopulation interactions, we have devised a method to determine the subpopulation composition of tumours by using paired tumour cell lines able to grow in different selective media. Line 4T07 forms colonies in thioguanine but not in HAT and line 168 forms colonies in HAT but not in thioguanine. An independent technique of determining tumour cell content was used to validate this method: line 168 and 4T07 cells are distinguishable by flow cytometry after staining with propidium iodide for DNA content. Mixtures of cell suspensions prepared from each unmixed tumour, as well as from tumours arising from mixtures of these lines, were analysed by both the colony formation assay and by the DNA content assay. The colony formation assay yielded values in good agreement with the DNA content assay, but was considerably more sensitive in that it was able to quantitate minority subpopulations that constituted less than 10% of the tumour. Both methods revealed that in tumours arising from mixtures, the tumour cells were almost entirely line 4T07, even when the inoculum had contained a high proportion of 168 cells. Since line 168 cells are very tumorigenic per se, these results suggest that line 4T07 cells are capable of interfering with 168 proliferation in mixed tumours, either directly or through a host-mediated mechanism.


Improvements to the original procedure of using a panel of monoclonal antibodies and magnetic microspheres for the depletion of tumour cells from bone marrow are described. These include a completely disposable system for the magnetic depletion of tumour cells coated with magnetic microspheres. Properties of a new series of microspheres are compared with the old M330 beads in their ability to deplete neuroblasts from both model systems and 50 bone marrows harvested from Stage IV neuroblastoma patients. Using human neuroblastoma cell lines labelled with the DNA intercalating, Hoechst dye 33342 a 5% tumour contamination can routinely be removed from 5 X 10(6) - 5 X 10(7) nucleated cells. Analysis of the 50 purged marrows revealed that 10 were visibly contaminated with tumour . In all but one case, tumour cells were removed. In this instance the tumour:bead ratio fell to 1:4 indicating the importance of maintaining a sufficient number of beads in the system. Red cell contamination of marrow was also kept extremely low so preventing possible physical blockade of bead:tumour cell interaction. Marrow engraftment was rapid in this group, apart from patients who had been exposed to high doses of alkylating agents prior to autografting.


Antibodies directed against tumour associated antigens provide a means for delivering preferentially cytotoxic radionuclides to the cells of primary and secondary tumours. The factors that influence the effectiveness of the radiation in the tumour compared with its effect on the radiosensitive normal tissues include the specificity of the antibody, the distribution of targeted energy within the tumour and the host's response to the injected foreign antibody. Recently some encouraging results from clinical trials of radioimmunotherapy have been reported in the literature. There is a continual search for more avid and specific antibodies, and the techniques of genetic engineering are being applied to the problem of reducing the antigenicity and mass of the carrier antibody. The improved efficiency of the labelled antibody needs to be supplemented by an identification of those tumours most likely to respond to this form of therapy.


Such animals show extensive loss of body weight, wasting of the muscle and adipose tissues, hypoglycaemia, and hypercalcaemia, even when the tumour weight comprises only about 1.9% of carcass weight. In contrast, the same tumour when inoculated into the liver does not cause any sign of tumour-related cachexia even when the tumour becomes much larger (6.6% of carcass weight). Interleukin 6 (IL-6), a mediator associated with cachexia in this tumour model, is detected at high levels both in the tumour tissues and in the circulating blood of mice bearing colon 26 tumour at the s.c. inoculation site. In contrast, only minute levels of IL-6 are detected in the tumour grown in the liver. The colon 26 tumour grown in the liver does not lose its ability to cause cachexia, because the tumour when re-inoculated s.c. is able to cause extensive weight loss and produce IL-6 as did the original colon 26 cell line. Histological studies revealed differences in the composition of tumour tissues: the tumours grown in the subcutis consist of many polygonal tumour cells, extended-intercellular space, and high vascular density, whereas those grown in the liver consist of spindle-shaped tumour cells. Thus, the environment where tumour cells grow would be a critical factor in determining the cachectic phenotype of cancer cells, including their ability to produce IL-6.


Shortly after grafting of Ehrlich ascites carcinoma cells, the serum of tumour-bearing mice loses the capacity to mediate immunoadherence phenomena, because of a sharp decrease in the concentration of C3b and C3d, while the cellular receptors for such factors are unaffected by tumour growth. It is suggested that complement is consumed through the alternative pathway which is activated during the inflammatory responses accompanying tumour growth.


The growth potential of a tumour can significantly depend on host features such as age, cell proliferation rates and caloric intake. Although this is widely known, existing mathematical models for tumour growth do not account for it. We therefore developed a new model for tumour growth, starting from a mathematical framework that describes the host's physiology. The resulting tumour-in-host model allowed us to study the implications of various specific interactions between the energetics of tumour and host. The model accounts for the influence of both age and feeding regimen of the host organism on the behaviour of a tumour. Concerning the effects of a tumour on its host, it explains why tumour-mediated body-weight loss is often more dramatic than expected from the energy demands of the tumour. We also show how the model can be applied to study enhanced body-weight loss in presence of cachectic factors. Our tumour-in-host model thus appears a proper tool to unite a wide range of phenomena in tumour–host interactions. Their use to describe data on tumours growing in vivo may be less warranted because of interactions between tumour and host. The aim of this article is to develop a mathematical model to explore such interactions between the growth of a tumour and the physiology of the host organism.Mathematical models for tumour growth have been widely used in different subdisciplines, such as cancer risk assessment , cancer We based our model on well-recognised interactions between tumour growth, energy homeostasis, utilisation of stored energy by tumour and host and cancer cachexia. The formulation in terms of a mathematical model has several benefits. First, it forces us to specify quantitative formulations about the interactions, which improves testability of the hypotheses involved. Second, because the model asks for an overall view of a number of processes and their inter-relationships, it can offer insights that complement those arising from individual experimental studies. Finally, model simulations allow to switch on or off particular hypothetical mechanisms easily, so that we can evaluate their impact on and relevance for the expected outcome.vice versa. Regarding the influence of the host on tumour behaviour, we focus on the implications for the tumour of differences in host energetics associated with host age and host caloric intake. Thereafter, we study the decrease in body weight associated with the increase in size of a tumour. Finally, we discuss several implications of the results obtained. The Appendix contains additional information on the mathematical formulation of the model as well as on the fitting procedures and parameter values.The article is organised as follows. First, we introduce the dynamic energy budget (DEB) theory ,2001, whTo model the interaction between tumour and host, we need a general framework describing the physiology of the host organism. Such a framework is provided by the DEB theory. The theory starts with a set of rules to characterise an individual organism, based on fundamental mechanisms that all organisms seem to have in common. From these rules, the theory derives quantitative expressions for sundry physiological processes. In this article, we explain only those aspects of the theory indispensable to understand our model for tumour growth. A more complete, though still qualitative, introduction to the theory can be found in V(t). Both body components have, by assumption, a constant, but not necessarily identical, chemical composition. As the relative amount of reserves and structure can vary, the composition of the total body can vary. For instance, during fasting the body loses predominantly reserves, so that the overall composition of the body changes.Figure 1M], are constant, which implies that total maintenance costs amount to M(t)=[M]V(t) per time unit. This assumption leads to a relationship between body size and respiration that accounts for both growth and maintenance. This prediction is well supported by experimental data concerning the scaling of respiration with body size  and reproductive processes (development and reproduction) take place in parallel. This is supported by the observation that some species start reproduction while they are still growing; others start reproduction well after reaching adult size. Yet in both species, growth levels off in the same way. This implies that the onset of reproduction cannot be the cause of the cessation of growth.κ-rule, an individual spends a fixed fraction κ of the available energy on somatic processes (growth and maintenance), whereas it spends the remainder fraction on reproductive processes . The part of the κ-rule concerning growth can be written asAccording to the so-called C(t) being the utilisation rate at time t; the utilisation rate is the rate at which energy is mobilised from the reserves and is made available for physiological processes =[G]dV/dt, with [G] being a constant. With the energy available for growth To stay alive, an animal has to give maintenance priority over growth. Increase in size consequently ceases when all energy available for maintenance and growth is spent on maintenance only. Maintenance thus determines the ultimate size an organism can reach. The costs of growth are the same for each unit increase in size. Thus, costs of growth per time unit are proportional to the increase in structural volume: C(t) (see The DEB model provides a quantitative expression for the utilisation rate C(t) see . When foC(t) see . This eqC(t) see .κ-rule above, we treated the animal's structure as a single variable. Since we want to describe tumour growth within the DEB framework, we have to expand the basic formulation. Suppose we zoom in on a cell that changes into a tumour cell. From an energetic point of view several things may happen. First, because tumour tissue is generally less differentiated than other tissues, tumour growth and maintenance costs per tumour volume may be lower, allowing tumour cells to proliferate faster than normal cells. However, because a tumour is a part of the body that has run out of control, a second energetic aspect may also change: a tumour cell may consume more than its share of the available energy, at the expense of other tissues. In other words, tumour cells may become gluttonous, taking what they want, and leaving the left-over available to the body proper. Thus, in our approach to tumour growth, mutations can lead to hyperplasia by decreasing the costs of somatic processes (maintenance or growth) or by increasing the energy supply per cell.In the introduction to the V, we consider tumour size Vu. Obviously, to survive and proliferate, the tumour has to obtain nutrients from the host. We characterise the gluttony of the tumour by a coefficient μu. If μu=1, each tumour cells demands the same amount of energy as an average normal cell; if μu>1, then a tumour cell takes more than an average body cell. Below, we will argue that the gluttony coefficient μu plays an important role in determining the aggressiveness of a tumour.To model tumour growth dynamics, we need some additional variables and parameters. In addition to body size κu(t) of the energy that the host has available for somatic processes. This assumption implies that tumours have priority for the resources over the host, which is supported by experimental evidence , rather than the utilisation rate itself, is important for tumour growth. As can be seen from Figure 3C] at the beginning. After some time, however, the body adapts to low food availability and the difference in [C] with food availability disappears. This is in agreement with the experimental observation that differences in energy expenditure per lean body mass disappear with long-term caloric restriction . Moreover, because tumour cells have no fine-tuned morphology, it seems likely that tumour growth costs are less than host growth costs . The same logic applies to tumour maintenance costs .In addition to the choice of the rat physiological parameters values, we also need to characterise the tumour by choosing appropriate parameter values. Because of the lack of adequate tumour growth data, we choose these values with an eye on host parameter values. Basically, three parameters characterise the tumour: its coefficient of gluttony a priori assumptions on the shape of the tumour growth curve. Our simulations show that both saturating and nonsaturating growth patterns are possible  of the same type of tumour in both animals. The resulting tumour growth patterns are shown in Figure 5per se, the predicted age-related differences in tumour growth can be attributed to changes in the energetic state of the host during its lifespan. vs young host, leading to slower tumour growth in the older hosts.To study the effect of host age on tumour progression with our modelling approach, we considered two vely see . We simuAnother aspect of tumour–host interactions is the effect of host nutrition on tumour growth. In the context of the DEB theory, physiological processes such as energy expenditure, body growth, and ageing depend on food intake  reduce its own maintenance investment and (b) degrade structural biomass. The former entails that not all required maintenance processes are carried out, which may lead to serious physiological problems and predispose for disease. The latter results in loss of, for instance, skeletal muscle, which may ultimately lead to death.κ-rule (equations (3)) allows for body-weight loss, there are two reasons why it would be inappropriate to use these equations to describe tissue degradation. First, if these equations were used, all energy originally invested in ‘building’ a unit biomass would be regained, which is thermodynamically impossible. Second, equations (3) imply that the host reutilises all energy released from tissue degradation to pay its own maintenance costs. This contradicts accepted knowledge, indicating that both host and tumour benefit from the released resources. We therefore have to account explicitly for tumour-mediated body-weight loss.Although the generalised κ-rule =−ω[G]dV/dt, which means that the amount of energy that becomes available per time unit is proportional to the tissue degradation rate . The parameter ω is an efficiency coefficient. The thermodynamic upper limit ω=1 means 100% efficiency, which, however, can never be achieved. In the realistic case that ω<1, part of the degraded structural biomass is actually wasted. Figure 7where ts, is indicated with a vertical line. Notice that total body weight . If σu>0, the cachexia-mediated degradation of structural biomass results in an energy release rate of Sc=ω[G]σuVu. In contrast, if σu=0 the tumour lacks any cachectic potency and the expressions above reduce to where Figure 8Above we argued that body-weight loss depends on the host physiological parameters e.g., . The samvice versa.The main difference between our approach and previous modelling approaches to tumour growth is that a tumour is conceived as a body part of the host rather than as an independent entity with an intrinsic maximum size. Our approach has the advantage that it can be used not only to describe tumour growth, but also to explore the relevance of interactions between tumour and host. We exemplified this by studying the influence of several host features on tumour behaviour and a priori the existence of an asymptotic maximum tumour size. In contrast, for the widely applied Gompertz model (a priori the existence of a maximum tumour size. Such an assumption hinders the possibility to predict under what physiological conditions a saturating tumour growth can be expected, and how the maximum tumour size depends on host and tumour characteristics.Another difference between our approach and others is that it does not assume tz model , maximumtz model . That isMu]=μu[M]. As this condition concerns tumour and host parameters, the shape of the tumour growth curve is determined by the energetic characteristics of both tumour and host.We analysed the relation between shape of the tumour growth curve and the parameters of the host. Existence of a maximum tumour size is only expected for tumours whose maintenance costs and capacity to extract nutrients from blood satisfy the condition [Various factors known to affect tumour growth are not accounted for by our model, for example, diffusion-limited nutrient availability, immune response or the presence of growth inhibitors. The main reason for this is that when multiple determinants of tumour growth are incorporated at once, it is very difficult to pinpoint the impact of any determinant in particular. Our approach allowed us, for instance, to show that tumour–host interactions in energy dynamics may already cause tumour growth to saturate. This implies that diffusion-limited nutrient availability may be sufficient e.g., , but notThere is general agreement about the main causes of age-dependency of cancer incidence. However, this does not hold for the mechanisms underlying age-dependent tumour progression. Among the mechanisms proposed to explain the latter phenomenon are changes in angiogenic capacity (We carried out a theoretical caloric restriction study to investigate the dependence of food consumption on a tumour's growth capacity. Model simulations suggested a strong dependence if tumorigenesis occurs after short-term caloric restriction. In contrast, a weak dependence of tumour growth on caloric intake is expected if tumorigenesis takes place after long-term exposure to caloric restriction. The dependence of tumour growth on food consumption can be understood on the basis of changes in the host energy expenditure.With regard to the influence of a tumour on host physiology, we focused on tumour-mediated body-weight loss. Computer simulations revealed that body-weight loss cannot be unequivocally linked to the increase in tumour size. The main reason is that the severity of body-weight loss is determined by the energetics of both host and tumour, rather than by the tumour alone. Moreover, part of the energy released is actually wasted. These model outcomes may well explain the observation by To illustrate the clinical utility of our model, we applied it to understand the energetics behind cancer cachexia. From an energetic point of view, cachexia involves several metabolic alterations, among which are an increase in energy expenditure, a decrease in both structural biomass and reserves, and a reduced food consumption. As a result, the host is maintained in a negative energy balance. In the context of our modelling approach, diminished food consumption is a consequence rather than a cause of body-weight loss in cachexia. Yet, in response to the decreased food intake, an acceleration of body-weight loss occurs. From the obtained model predictions, we conclude that the extent of the disease as well as the time delay between tumorigenesis and disease onset strongly depend on the physiological features of the host.A promising line of research would be to extend the model to include clinical interventions intended to reverse body-weight loss in tumour-bearing patients, such as food intake manipulations and parenteral nutritional support. Food intake manipulations can be incorporated, for instance, as an increase in the assimilation rate. Several authors discussed the possible benefits of a low-fat dietary intervention in cancer patients (


Using a microassay for cellular immunity, tumour specific cytotoxicity was detected in 2/5 cases of ocular melanoma and 1/3 cases of primary cutaneous melanoma before treatment. Reactivity was measured against allogeneic skin melanoma target cells in short or long term in vitro culture. Lymphoid cells from patients with disseminated cutaneous melanoma were either non-reactive (4/8 cases) or gave a nonspecific cytotoxicity on target cells of diverse histogenic origins. Among tumour-free patients tested after surgery, 0/2 patients with ocular tumour were non-reactive 3-4 months post surgery. After sugical excision of cutaneous melanoma 2/2 patients gave tumour specific reactions during the first month after surgery. After longer time intervals, from 5 months to 3 years, only 1/8 patients were reactive. Preoperative radiotherapy in a total skin dose of 10,000 rad produ-ed a transient tumour specific reaction 24 h after therapy in a single case. Following local tumour excision in patients given preoperative irradiation, 2 cases which had previously demonstrated tumour specific CMI lost reactivity. Among 14 tumour-free individuals tested only after preoperative radiotherapy and surgery, at intervals from 5 day to 13 years, a single case gave tumour specific CMI. Palliative irradiation in doses 4000-4960 rad to the inguinal or axillary lymph nodes was found to induce a generalized lymphopenia within 48 h after treatment. Lymphoid cell preparations from patients with localized melanoma contained significantly increased numbers of immature cells (lymphoblasts and myeloblasts) and myeloid precursor elements. Those prepared from patients with disseminated disease had in addition elevated levels of eosinophils but reduced numbers of recoverable lymphocytes.


To establish whether loss of heterozygosity (LOH) for chromosome 16q in Wilms' tumours confers an adverse prognosis, DNA from 40 Wilms' tumour/normal pairs were analysed using highly polymorphic microsatellite markers along the length of 16q. Fifteen per cent of tumours showed LOH for 16q. Although the common region of allele loss spanned the 16q24-qter region, a second distinct region of LOH was identified in 16q21. Five out of six tumours showing LOH were either (1) high stage or (2) low stage with unfavourable histology. In addition, there was a higher mortality rate in patients showing LOH for 16q than those that did not. These data strongly support the suggestion that LOH for 16q is associated with an adverse prognosis.


The synthesis and localization of collagen have been studied on material from a total of 16 primary Ewing's tumours. The predominant collagen extracted from the tissues and synthesized in short-term cultures was type I. The proportion of type III collagen was relatively small and variable (0-8%) in the direct tumour extracts, but a higher proportion  was synthesized in culture. Immunofluorescence studies showed that positive staining for all types of collagen tested  was restricted to stroma; there was no evidence of collagen either within the tumour cells or in their pericellular matrix, a finding endorsed by negative staining for reticulin in the same areas. The absence of any evidence for type IV or V collagen synthesis by Ewing's cells argues against an endothelial origin for the tumour, and indicates that collagen analysis is unlikely to be of value in the diagnosis of this particular sarcoma.


Mycobacterium butyricum gave better transplant immunity to tumour cell challenge than tumour extracts alone. Mice immunized with Mycobacterium butyricum alone prior to challenge with tumour cells, did not show any significant difference in the incidence of tumours from control mice.Transplant immunity to adenovirus 12-induced tumour cells was demonstrated in CBA mice which had been previously immunized with extracts of homologous tumour cells. Immunization of mice with tumour cell extract together with heat-killed


Patient. A case of peripheral leiomyosarcoma presenting with features of pulmonary thromboembolism is described.Discussion. Persistence of the embolus despite triple-armed thrombolytic therapy and the presence of intravascular tumour invasion suggest the rare entity of pulmonary tumour embolism from a leiomyosarcoma.


The levels of tumour-associated trypsin inhibitor (TATI), CA 125 and CEA were measured in ovarian cyst fluids from 21 patients. TATI in cyst fluid was immunologically and physicochemically similar to the peptide originally isolated from the urine of a patient with ovarian cancer. Mucinous cysts contained significantly higher levels of TATI than did serous cysts. Immunohistochemically TATI was localized in the apical parts of cells of mucinous ovarian cysts. These results suggest that this tumour-associated peptide is actually produced by a tumour. Like TATI, CEA occurred at higher concentrations in mucinous than in serous cyst fluids, whereas CA 125 was found in higher concentrations in serous than in mucinous cyst fluids. The concentrations of these tumours markers in cyst fluids did not correlate with circulating levels of the same markers. In spite of the very high levels of all these tumour markers in benign cyst fluids, serum levels were normal or only slightly elevated. Clearly elevated serum levels occurred only in patients with malignant tumours. Cyst fluid levels of these tumour markers could not be used to distinguish between benign and malignant tumours.


The authors also acknowledge support for this work from National Institutes of Health grant AI48489.


The presence of fibrin monomers in tumour cell injected animals suggested intravascular fibrin deposition; the plasma fibrinogen level, however, did not decrease significantly. Isotope studies using 51Cr labelled platelets revealed a rapid disappearance of the platelets from the circulation and their trapping in the lung—the primary site of tumour cell lodgement. Dipyridamole and ancrod pretreatment did not influence the decrease of platelets and their accumulation in the lung after tumour cell injection. In contrast, heparin completely prevented the thrombocytopenia and the platelet trapping in the lung. From the present experiments it is concluded that embolic tumour cells lead to early endothelial damage, resulting in local thrombin formation with subsequent irreversible platelet aggregation.After the intravenous injection of Walker 256 tumour cells into rats the platelet count decreased rapidly and remained low during the following period of observation. The platelet decrease was closely related to the number of cells injected. Intra-arterial tumour cell injections required a considerably higher tumour cell count to produce a comparable thrombocytopenia. Non-viable tumour cells and tumour cell fragments induced a similar decrease of circulating platelets. Neither viable tumour cells nor tumour cell fragments aggregated rat platelets


P=0.031). Expression of p21 and Mdm-2 did not correlate with the outcome of RT.The primary aim of this study was to investigate if the expression of the DNA damage identifying protein DNA-PKcs known to be involved in DNA repair after treatment with ionising radiation can be used as a predictive marker for radiotherapy (RT) response in cervical cancer. Formalin-fixed primary tumour biopsies from 109 patients with cervical cancer, FIGO-stage IB–IIA, treated with preoperative brachytherapy followed by radical surgery were analysed by immunohistochemistry. In addition, correlation studies between early pathological tumour response to radiation and expression of Ku86, Ku70, Mdm-2, p53 and p21 in primary tumours were also performed. We found that tumour-transformed tissue shows positive immunostaining of DNA-PKcs, Ku86 and Ku70, while non-neoplastic squamous epithelium and tumour-free cervix glands show negative immunoreactivity. Expression of DNA-PKcs positively correlated with both Ku86 and Ku70, and a statistically significant correlation between the Ku subunits was also found. After RT, 85 patients demonstrated pathologic complete remission (pCR), whereas 24 patients had residual tumour in the surgical specimen (non-pCR). The main finding of our study is that there was no correlation between the outcome of RT and the expression of DNA-PK subunits. Positive p53 tumours were significantly more common among non-pCR cases than in patients with pCR ( Early stage cervical cancer can be successfully treated by radiotherapy (RT). We have shown that pathologic complete remission (pCR) after preoperative brachytherapy as compared to incomplete pathologic remission (non-pCR) is a strong predictive factor for long-term survival  are considered as the most lethal lesions and sensitivity to ionising radiation will depend on the DSB repair efficacy possessed by the cell. Nonhomologous end joining (NHEJ) is a major pathway for repair of DNA DSBs in mammalian cells . The iniin vitro, including the transactivation domain of p53  staining providing us with a frequency of stained tumour cells for each case analysed. These data were correlated with treatment response defined as pathological complete tumour remission after RT.Our primary end point was pathological complete tumour remission. Our hypothesis was that low percentage immunostaining (below the median) of DNA-PKcs in primary tumour would result in pCR after RT to a greater extent than high (above the median) immunopositivity of DNA-PKcs. Our null hypothesis was that no difference in pathological complete tumour remission prevailed in that respect. If the patients with low DNA-PKcs showed a rate of complete pathological tumour remission of 45% and the ‘high’ group had 20% complete tumour remission we should need 2 × 43=86 patients to detect that difference. This is true if the significant level is 5%, the power is 80% and with a one sided hypothesis.−1. In all, 40 patients were treated with the manual technique using radium applicators with a calculated total uterovaginal mean dose of 6500 milligram-hours of radium (mghRa) and a retrospectively estimated dose rate to point A of 1.1 Gy h−1. Tumour response did not differ significantly between the two techniques. The use of biopsies and surgical specimens for the present study was approved by the Ethics Committee at the Karolinska Institute. Informed consent were received from all patients alive at time of analysis.Pretreatment biopsy specimens were obtained from 109 patients with cervical carcinoma FIGO stage IB-IIA, treated at the Department of Gynaecologic oncology, Radiumhemmet, during January 1989 to December 1991. Age at time of diagnosis varied between 23 and 75 years, with a mean age of 44 years. Median follow-up for patients alive and not alive were 69 months (range 50–109 months) and 19 months (range 0–87 months), respectively. There were 25 patients with small tumours (<2 cm), 47 patients with large tumours (⩾4 cm) and 37 patients had tumours of intermediate size (2–<4 cm). Tumour characteristics, such as stage distribution, tumour size, histopathological type and grade in relation to the degree of tumour remission after RT is shown in Evaluation of RT response was performed by histopathological examination of the formalin-fixed surgical specimen within 1 week after surgery. All specimens were re-evaluated by a senior pathologist. Tumour remission was assessed in the surgical specimen and was classified as pCR if no morphological intact tumour cells were found or as non-pCR if residual tumour was detected.μm), placed on Super Frost Plus slides, were dewaxed and rehydrated. The slides were boiled in citrate buffer pH 6.0, in a microwave oven for 12 min at 800 W and 20 min at 250 W. The slides were then cooled and rinsed with phosphate buffer saline (PBS). After blocking with 1% BSA, the proteins were detected by using mouse monoclonal antibodies to DNA-PKcs 1 : 100, Ku86 1 : 200, Ku70 1 : 200 , p53 1 : 100 , p21 1 : 50 , and Mdm-2 at 1 : 50 . The protein primary antibody was then detected by incubation with a biotinylated secondary antibody and ABC complex (Vector Laboratories), using diaminobenzedine substrate as a chromogene. The specimens were counterstained with haematoxylin. Staining evaluation was performed in a blinded fashion, without prior knowledge of the clinical data or outcome for the individual patient. Counting was carried out on four representative areas in a light microscope. In every specimen 500 tumour cells were counted and the percentage of positive stained cells was determined. Intensity of immunostaining was judged as low, intermediate or strong. Morphologically normal cells in each specimen served as internal negative control. The intraobserver reproducibility was tested and showed significant correlation between the two series of evaluations .Immunostaining was preformed using the ABC-technique . Paraffin sections  was calculated to compare expression of proteins. Survival analyses on continuous background factors were calculated by Cox regression bivariate analysis.Statistical tests on data were based on Pearson correlation test was used to test intraobserver variation.rs=0.49, P<0.001) as well as with Ku70 positivity . In addition, a correlation between the Ku subunits  was found.Immunostaining was performed on formalin-fixed biopsies against DNA-PKcs , Ku86 F and Ku70rs=0.27, P=0.005) as well as between p53 and Mdm-2  When the frequency of p53 positivity was compared between small/intermediate tumours and large tumours, the amount of cells staining positive for p53 was found to be significantly higher in large tumours (P=0.018). In addition, a positive correlation between tumour size and immunopositivity for p21 was also found (P=0.002). No correlation could, however, be found between tumour size and high and low expression of Mdm-2. p53-positive tumours showed a significantly higher expression of DNA-PKcs than p53-negative tumours (P=0.009). No significant difference in protein expression was detected between the different histogenetic types of cervical carcinoma. In addition, differences in protein expression between grades of differentiation could not be found. No correlation was found between long-term survival and expression of any of the analysed proteins   or high percentage stained cells . We could not detect any significant difference in protein expression of DNA-PKcs in primary tumour between pCR and non-pCR cases. In addition there were no significant difference in protein expression of the subunits Ku70 and Ku86 between pCR and non-pCR cases . CompariP=0.031) . DiffereThe aim of our study was to see if there is a relation between DNA-PKcs and response to RT in homogenously treated cervical carcinoma of stage IB-IIA as assessed by IHC in the primary tumour. We were, however, not able to demonstrate such a correlation. In addition, related proteins as Ku70 and Ku86 also did not correlate with RT response. Among down streams effectors analysed like Mdm-2, p53 and p21 only the frequency of IHC determined p53-positive cells showed a statistically significant correlation with response to RT measured as pathological complete tumour response.γ-H2AX are constitutively activated and expressed at higher levels during early tumorigenesis as compared to normal tissue  of explanted tumour cells and expression of Ku70 or Ku80 in primary biopsies. A significant higher survival rate was found for patients whose tumours had a low level of Ku70 expression but no relation was found with low Ku80 expression. In contrast to Wilson's study, our study only included patients with early stages (IB–IIA) of cervical carcinoma. We did not find a correlation between DNA-PKcs and their subunits with response to RT. In addition, we did not find a correlation between expression in primary tumour biopsies and long-term survival for any of the studied proteins.The absence of a correlation between IHC measured expression of DNA-PK and tumour response is at some variance with other reports indicating a positive role of DNA-PK as a predictive marker for radiosensitivity in cervical carcinoma. In a study by in vitro methods such as soft agar clonogenic assay could be questioned since methodological factors may have a considerable effect on the SF2-values of explanted tumour cells which has to be accounted for when the results are interpreted. Using survival as an end point when evaluating molecular factors of importance for clinical response to RT, also has drawbacks. Survival is a variable that strongly is affected by tumour stage, age, performance status, intercurrent diseases and all treatment given to the patient. The comparison of tumour response to expression of DNA-PK seems less biased.Differences in patient populations concerning tumour stages may be of importance for diverging results since it is not clear if or how the molecular pattern in tumours changes with progressive disease. In addition, the definition of response to RT differs between studies. As surgery was included in the treatment schedule in our study we based the definition of treatment response on the pathological evaluation of the whole cervix. Defining response to RT in the individual patient by vs 44 in our study) and also by that a major part of patients presented more advanced stages (stage III–IV). A radioresistant phenotype of a tumour may originate from a small population of cells possessing the prerequisite for survival after irradiation, indicating that heterogeneity of level of protein expression on the cellular level may be of importance when searching for markers that can predict tumour response. In the Harima study the IHC scoring system differs from ours with the inclusion of staining density, a parameter which we have left out. In our study, no differences in intensity of staining could be detected for any of the analysed proteins when comparing pCR and non-pCR samples.in vitro radiosensitivity of explanted tumours (When assessing molecular factors important for response of tumours to RT it is important to consider the impact of FIGO-stage and tumour size (Prognostic significance for p53 immunostaining has previously been evaluated for RT in patients with cervical carcinoma (In conclusion, results of the present study suggest a role for DNA-PK in tumour transformation since elevated levels of DNA-PKcs, Ku86 and Ku70 were found in tumour tissue as compared with non-neoplastic tissue. Our results, however, imply that immunohistochemical assessment of the DNA-PK complex cannot be used as a predictive marker for clinical response to brachytherapy of early stages of cervical carcinoma. However, expression of p53 protein is more frequent among cases with incomplete pathological remission after brachytherapy of cervical carcinoma.


Antibodies are thought to bring about tumour cell lysis by antibody-dependent cell-mediated cytotoxicity (ADCC), but the exact mechanism is not well elucidated. Monocytes are known to be important mediators of anti-tumour ADCC and are also known to secrete the cytokines tumour necrosis factor alpha  and interleukin 1 beta (IL-1 beta), both of which have been shown to bring about tumour cell lysis. We examined the release of these cytokines during ADCC and attempted to elucidate which components of the ADCC reaction were necessary for cytokine production. We measured TNF-alpha and IL-1 beta in supernatants collected from a standard ADCC assay using each of the anti-colorectal antibodies m17-1A, c17-1A and cSF25. We found that there was significant TNF-alpha and IL-1 beta release during ADCC mediated by each of these three antibodies and that the magnitude of cytokine release seemed to reflect the degree of tumour cell lysis produced by each antibody. Furthermore, we found that effector cells, target cells and a specific anti-tumour antibody were necessary for this to occur. The presence of only some of the components of the reaction or of an irrelevant antibody produced little or no TNF-alpha or IL-1 beta. We conclude that TNF-alpha and IL-1 beta are released when an effector and tumour target cell are united by a specific tumour antibody and that these cytokines may be important in bringing about tumour cell lysis during the ADCC reaction.


Novel knowledge on this compartment may provide important insight into how tumors develop and how they respond to therapy.The interstitium, situated between the blood and lymph vessels and the cells, consists of a solid or matrix phase and a fluid phase, together constituting the tissue microenvironment. Here we focus on the interstitial fluid phase of tumors, We discuss available techniques for interstitial fluid isolation and implications of recent findings with respect to transcapillary fluid balance and uptake of macromolecular therapeutic agents. By the development of new methods it is emerging that local gradients exist in signaling substances from neoplastic tissue to plasma. Such gradients may provide new insight into the biology of tumors and mechanistic aspects linked to therapy. The emergence of sensitive proteomic technologies has made the interstitial fluid compartment in general and that of tumors in particular a highly valuable source for tissue-specific proteins that may serve as biomarker candidates. Potential biomarkers will appear locally at high concentrations in the tissue of interest and will eventually appear in the plasma, where they are diluted.Access to fluid that reliably reflects the local microenvironment enables us to identify substances that can be used in early detection and monitoring of disease. The interstitial space consists of connective and supporting tissues of the body and is located outside the blood and lymphatic vessels and parenchymal cells. Essentially the interstitium can be divided into two compartments: the interstitial fluid and the structural molecules of the interstitial or the extracellular matrix (ECM). In the present review we focus on the interstitial fluid phase, which contains an array of regulatory molecules defining the physical and biochemical microenvironment of cells. All organs have an interstitium, although at a variable amount. The structure and composition of the interstitium differs considerably depending on the mother organ. Interstitial water with its solutes, the interstitial fluid volume, serves as a transport medium for nutrients and waste products between cells and capillary blood and also harbors various signaling substances that are either produced locally or brought to the organ by the circulation.Recently there has been a renewed interest in the tumor microenvironment because of its role in tumor growth and metastasis. The tumor microenvironment can be defined as the insoluble elements of the ECM, the stroma with its cellular elements such as fibroblasts and immune cells and the fluid phase of dissolved substances. Traditionally the focus has been on the stroma and the cellular elements of the tumor, whereas here we focus on the fluid phase that may be thought of as a "misconsidered component of the internal milieu of a solid tumor" .We discuss recent data on the isolation of interstitial fluid and what can be learned from analyses of such fluid regarding local production of signaling substances and potential biomarkers as well as the level of interstitial fluid colloid osmotic pressure, one of the determinants of transcapillary fluid exchange. For a broader description of the latter topic, the reader is referred to reviews on transcapillary fluid exchange and interstitial fluid volume regulation -9. The ci.e., an extracellular or interstitial matrix composed of solid elements constituting the framework of the tumor and a fluid phase constituting the interstitial fluid; it has its special features that are discussed briefly here. Before we turn to the interstitial/extracellular fluid phase, we summarize some common features of the ECM or solid phase in tumors compared to normal interstitium, as schematized in Figure e.g., , as re e.g., . These cre e.g., .e.g., [i.e., the fluid bathing the tumor and stroma cells, is of considerable importance to understand how tumors develop and progress. The properties of the tumor interstitium, e.g., the high vascularity and cellularity, represent major challenges when trying to gain access to the TIF.The abnormal interstitium representing the microenvironment of the tumor cells has puzzled researchers for many years and continues to do so. Recently, an increased interest in the tumor microenvironment and its influence on cancer progression has turned attention toward this compartment  or simultaneously with tumor implantation (termed chronic wicks) and characterized the isolated fluid. In addition to evaluating the effect of acute and chronic insertion, they evaluated the effect of both implantation time and tumor type. They concluded that acute wick implantation requires a long time for equilibration  and that chronic wicks should be preferred to avoid bleeding and cellular damage. Furthermore, as pointed out by Stohrer et al., cellular proteins deriving from necrotic cells or cells disrupted during wick removal may enter the wick fluid and cause a higher protein concentration (and higher colloid osmotic pressure). As a result, the protein distribution pattern will deviate from that of undisturbed tumor interstitial fluid, i.e., mainly proteins with lower molecular weight (MW) than albumin, e.g., as observed in skeletal muscle [As for skin and muscle , wicks et al.  implantel muscle .e.g., [The microdialysis technique, based on diffusion of analytes across a semipermeable membrane, has been applied extensively to study TIF . Wite.g., ,55). In Ultrafiltration has traditionally been used for separation or purification of chemicals, but this technique has also been applied to sample tissue fluid by implanting capillary ultrafiltration probes in vivo (reviewed in ). With t51Cr-ethylenediaminetetraacetic acid (EDTA) as a probe to show possible 'contamination' of cellular fluid. Addition of tumor cell fluid to the centrifuged volume should thus show up as a reduced 51Cr-EDTA concentration in the centrifugate relative to plasma  that were washed carefully and placed in tubes containing phosphate-buffered saline. After incubation or elution for 1 h followed by centrifugation, the supernatant was collected and named tumor interstitial fluid. Later, they used the same elution method to isolate fat interstitial fluid [Recently, in a search for novel biomarkers, Celis et al.  proposedal fluid . In the The functional importance of the TIF has been acknowledged in earlier studies by Jain , and it et al. [+, CO2, and lactic acid and low glucose and O2, probably a result of tumor metabolism [CO2 was 16-39 mmHg higher, and the concentration of bicarbonate was 4-6 mmol/l higher when compared to afferent plasma.Gullino et al. ,69 were tabolism . Of notetabolism , the PCOi.e., by the net filtration pressure being the difference between the hydrostatic and colloid osmotic pressure acting across the capillary wall.There has, however, been some development during recent years regarding the macromolecular composition of TIF that is of importance for our understanding of transcapillary fluid balance in tumors. The transcapillary fluid exchange is determined in tumors as in normal tissue by the Staling principle, et al. [TIF in four different human tumor xenografts in mice using implanted wicks. Using chronic wicks (see section "Implanted wicks" above), they found a generally higher pressure in TIF than in subcutaneous tissue. In three of the four tumor types, COP in TIF was not significantly different from plasma , whereas in a colon adenocarcinoma (LS174T), the COP was 82% of that in plasma and the COP in subcutaneous interstitial fluid was 41% of the plasma value. In a recent study, we measured COP in TIF isolated by centrifugation of excised chemically induced mammary carcinomas [et al. [et al. [Whereas there are numerous studies where tumor interstitial fluid pressure has been measured and found to be elevated ; in twoet al.  determinrcinomas , and we  [et al. , althouget al. [et al. [et al. [et al. [The observed protein distribution pattern in interstitial fluid may also be of interest since this pattern may have implications for the interpretation of potential biomarker candidates that are discussed in the following section. Gullino et al.  subjecte [et al.  after se [et al. , these s [et al. . We comp [et al. , and whe [et al.  discussee.g., using proteomic approaches, it is also important to quantify known bioactive compounds in the interstitial fluid. Numerous growth factors and cytokines are associated with and sequestered in the ECM . Pos. Pose.g.Dabrosin . In a maHaving demonstrated that interstitial fluid could be isolated from bone marrow in rats and humans by centrifugation , we thene.g. [i.e., in the tissue microenvironment. Tumor cells are known to release specific substances that disrupt the tissue and elicit local responses, and it is likely that candidates can be found locally in the tumor cell secretome, i.e., specific substances secreted by tumor cells [The advances of mass spectrometry techniques combined with bioinformatics allow detection, identification and quantification of numerous peptides and proteins in biological samples e.g. -86). The. Thee.g.e.g.  and may e.g. ). One gee.g. ,90. Stilor cells -94. For et al. [Although substantial interest has been devoted to the ECM, the same cannot be said about the interstitial or extracellular fluid phase. Here we have focused on the interstitial fluid phase of tumors, and although the importance of this type of study in normal as well as neoplastic tissues was emphasized more than 20 years ago , there het al.  more thaThe authors declare that they have no competing interests.HW conceived of the study and wrote the paper, OT, POI, RK and RB participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.


To demonstrate associations of certain chromosomal aberrations with defined renal cell tumour (RCT) subtypes, we analysed 239 tumour nephrectomy cases for specimens with multicentric tumours. Chromosomal in situ hybridization was then performed on 15 cases with 34 foci , and 18 papillary RCTs (11 carcinomas and seven adenomas) for specific chromosomal aberrations, using α-satellite probes for chromosomes 3, 7 or 17. Particular preference was given to cases which had separate foci with different cytomorphologies. Furthermore, we compared aberrations in relation to tumour size, stage, grade and between different foci in a specimen. Thirty-four cases had multiple tumours. Forty-seven per cent of the multicentric tumours were conventional RCCs and 53% papillary RCTs . Three conventional RCCs sized 8 mm (G3), 13 cm  and 15 cm , respectively, revealed monosomy 3, and 13 were disomic. Seventeen papillary RCTs (11 carcinomas and six adenomas) displayed trisomy 17, irrespective of size or grade. Four papillary carcinomas and six papillary adenomas had trisomy 7, and the rest (seven papillary carcinomas and one papillary adenoma) revealed disomy 7. In conclusion, papillary RCTs were tendentially multicentric. Although specific for conventional RCCs heedless of size, monosomy 3 was only observed in high-grade and/or advanced tumours. Trisomy 17 was only detectable in papillary RCTs irrespective of tumour state, showing increased copies with tumour growth. Papillary RCTs also appeared to lose some copies of chromosome 7 with tumour progress, possibly reflecting malignancy. © 2000 Cancer Research Campaign


A clear tumour image was obtained with 131I-labelled-HML-Fab at 3 h post-injection. The use of HML as a radiolabelling reagent, combined with angiotensin II treatment, efficiently improved tumour targeting and enabled the imaging of tumours. These results suggest the feasibility of PET scan using antibody fragment labelled with 18F-fluorine substitute for radioiodine. © 1999 Cancer Research CampaignImmunoscintigraphy is a tumour imaging technique that can have specificity, but high background radioactivity makes it difficult to obtain tumour imaging soon after the injection of radioconjugate. The aim of this study is to see whether clear tumour images can be obtained soon after injection of a radiolabelled reagent using a new linker with antibody fragments (Fab), in conditions of induced hypertension in mice. Fab fragments of a murine monoclonal antibody against human osteosarcoma were labelled with radioiodinated 3′-iodohippuryl N-ɛ-maleoyl-L-lysine (HML) and were injected intravenously to tumour-bearing mice. Angiotensin II was administered for 4 h before and for 1 h after the injection of radiolabelled Fab. Kidney uptake of


Transplanted syngeneic osteosarcomas (induced by 32P in DA rats) grew significantly larger in DA rats receiving a simultaneous transplant of allogeneic osteosarcoma than in rats receiving syngeneic tumour only (P less than 0-01). Two other malignant allogeneic tumours, and allogeneic spleen cells, did not alter the growth of the transplanted syngeneic osteosarcomas. When the allogeneic osteosarcoma was given 7 days before the syngeneic tumour, the reverse effect (retardation) occurred. When given 7 days after the syngeneic tumour cells, the effect on both syngeneic and allogeneic tumour growth was variable. Some possible reasons for these findings are discussed, and the argument is presented that immunological phenonema are involved in the reaction.


Nω-nitro-L-arginine methyl ester (L-NAME) or S-methylisothiourea sulphate (SMT). In contrast, HO-1 was found only in tumour cells, not in macrophages, by in situ hybridization for HO-1 mRNA. HO-1 expression in AH136B cells in culture was strongly enhanced by an NO (NO+) donor S-nitroso-N-acetyl penicillamine. HO-1 mRNA expression in the solid tumour in vivo decreased significantly after treatment with low doses of NOS inhibitors such as L-NAME and SMT (6–20 mg kg−1). However, the level of HO-1 mRNA in the solid tumour treated with higher doses of NOS inhibitor was similar to that of the solid tumour without NOS inhibitor treatment. Strong induction of HO-1 was also observed in solid tumours after occlusion or embolization of the tumour-feeding artery, indicating that ischaemic stress which may involve oxidative stress triggers HO-1 induction in the solid tumour. Lastly, it is of great importance that an HO inhibitor, zinc protoporphyrin IX injected intra-arterially to the solid tumour suppressed the tumour growth to a great extent. In conclusion, HO-1 expression in the solid tumour may confer resistance of tumour cells to hypoxic stress as well as to NO-mediated cytotoxicity. © 1999 Cancer Research CampaignInduction of haem oxygenase-1 (HO-1) as well as nitric oxide (NO) biosynthesis during tumour growth was investigated in an experimental solid tumour model (AH136B hepatoma) in rats. An immunohistochemical study showed that the inducible isoform of NO synthase (iNOS) was localized in monocyte-derived macrophages, which infiltrated interstitial spaces of solid tumour, but not in the tumour cells. Excessive production of NO in the tumour tissue was unequivocally verified by electron spin resonance spectroscopy. Tumour growth was moderately suppressed by treatment with either


This study has evaluated the effect of breathing 100% oxygen, carbogen and carbon monoxide (at 660 p.p.m.) on the bioenergetic and oxygenation status and the radiation response of 200-mm3 C3H mammary carcinomas grown in the feet of CDF mice. Bioenergetic status was assessed by 31P magnetic resonance spectroscopy (MRS) using a 7-tesla spectrometer with both short (2 s) and long (6 s) pulse repetition times. Tumour partial pressure of oxygen (PO2) was measured with an Eppendorf polarographic electrode; the oxygenation parameters were the median pO2 and fraction of pO2 values < or = 2.5 mmHg. The radiation response was estimated using a tumour growth delay assay (time to grow three times treatment volume). Carbon monoxide breathing decreased tumour pO2 and compromised the radiation response, but the beta-nucleoside triphosphate (NTP)/Pi ratio was unchanged. Both carbogen and oxygen (100%) increased tumour pO2 and beta-NTP/Pi and enhanced the radiation response, the effects being similar under the two gassing conditions and dependent on the gas breathing time. Thus, in this tumour model, 31P-MRS can detect hyperoxic changes, but because cells can remain metabolically active even at low oxygen tensions the beta-NTP/Pi did not correlate with low tissue oxygenation. An analysis of variance showed that gas breathing time induced a significant systematic effect on beta-NTP/Pi, the MRS pulse repetition time had a significant effect on beta-NTP/Pi change under hypoxic but not under hyperoxic conditions and the type of gas that was inhaled had a significant effect on beta-NTP/Pi.


For the cachexia-inducing MAC16 tumour, optimal anti-tumour activity was seen at dose levels of 10 and 25 mg kg-1 day-1, together with a reversal of cachexia and a doubling of the time to sacrifice of the animals through cachexia from 8 days to 17 days. The remaining tumour fragments showed extensive necrosis in regions distal from the blood supply. Growth of the MAC13 tumour was also effectively suppressed at dose levels between 5 and 50 mg kg-1 day-1, resulting in a specific growth delay between 1.0 and 1.2. Growth of the MAC26 tumour was also inhibited a concentration-related manner, with doses of 25-50 mg kg-1 day-1 being optimal. Anti-tumour activity towards all three tumours at low dose levels of CV-6504 was effectively suppressed by concurrent administration of linoleic acid (1 g kg-1 day-1), suggesting that inhibition of linoleate metabolism was responsible for the anti-tumour effect. Tumour sensitivity may be correlated with increased DT-diaphorase that are required to metabolise CV-6504 to the active hydroquinone, which inhibits 5-lipoxygenase activity.


Local injection (i.e. injection at the site of tumour inoculation) of strains of C. Parvum which have a significant anti-tumour effect when given systemically  strongly inhibits the growth of isogeneic transplants of a fibrosarcoma in intact CBA mice but has little or no effect on subcutaneous transplants of the same tumour in T-cell deprived mice. The anti-tumour effect of local injection of C. parvum, unlike that of systemic injection in this particular tumour system, thus appears to be T-cell dependent.


This may be one mechanism explaining the higher response rate of tumours in oestradiol-replenished mice.The majority of breast cancers are oestrogen dependent and although current treatment strategies have improved, approximately 50% of the patients will develop metastasis. New treatments that result in long-term systemic immunity are therefore being developed. We have previously shown that adenoviral gene transfer of B7-1/IL-2 to murine breast cancer induces a high rate of complete tumour regression and systemic immunity. Since oestrogens not only affect breast cancer but also have been shown to modulate immune function and secretion of immune-regulatory cytokines, we explored whether administration of oestradiol altered the immune response induced by an adenoviral vector expressing B7-1/IL-2. An oestrogen-dependent murine breast cancer tumour was used in ovariectomised mice, supplemented either oestradiol or placebo. We report the somewhat unexpected finding that intratumoral injection of adenovirus expressing B7-1/IL-2 induces complete tumour regression in 76% of oestradiol-supplemented mice, while only 18% of the tumours regressed in the oestrogen-depleted group. Cured mice in both groups exhibited a similar CTL response against the tumour antigen. However, intratumoral IFN- Breast cancer accounts for almost 30% of all cancer cases in women in the Western World and the incidence of the disease is still increasing  and B7-2 (CD86) are required for full activation of T cells. The B7 molecules are members of the immunoglobulin (Ig) superfamily and are ligands for CD28 and CTLA-4 counter-receptors on T cells  were purchased from Charles River . They were housed in pathogen-free conditions with a light/dark cycle of 12/12 h and fed with rodent chow and water β-oestradiol (E2), 0.18 mg per 60 day release, or placebo pellets  were implanted subcutaneously (s.c.) in the animal's back 7 days before tumour induction. The pellets provide continuous release of E2 at serum concentrations of 150–250 pM, confirmed by serum analysis assayed by Abbot Axsym Systems , which is in the range of physiologic levels seen in mice during the oestrous cycle (Mice were anaesthetised with isofluorane (Abbot), ovariectomised (OVX) and 3-mm pellets containing 17 us cycle .6 cells in 200 μl PBS were injected s.c. into the right hind flank of a syngenic female FVB/n host. Tumour volume was determined by measuring length, width, and depth of the tumour weekly using a caliper.Tumour cells were derived from a transgenic mouse strain expressing polyoma middle T (PyMT) antigen under the control of the mouse mammary tumour virus (MMTV) long-terminal repeat , and the control vector Ad3) animals were injected intratumorally with 1 × 109 PFU of AdB7-1/IL-2 or control Addl70-3 vector diluted (50 μl) in sterile endotoxin-free PBS and tumour growth determined weekly. In animals that showed complete regression of primary PyMT tumours, mice were rechallenged at 90 days with PyMT tumour cells by subcutaneous injection of 106 tumour cells on the opposite flank.Following tumour formation  .Specific PyMT antigen cytotoxic T-cell killing was measured as previously described . The sensitivity was 2 pg ml−1. All samples were analysed in duplicates and given results are the means of each duplicate.Tumours were removed, snap frozen in liquid nitrogen, and weighed 2 and 10 days after adenovirus administration. Tumours were homogenised in PBS containing 100 t-test and Fisher's exact test was used as appropriate. A P<0.05 was considered as statistically significant.Data are expressed as mean±s.e. m. All given values represent the mean of the duplicate analysis of each sample. Student's in vitro and tumour growth in vivo . Naive mice simultaneously injected with the same tumour cell suspension developed tumours within 21 days as expected. These results suggest that the cured mice in either E2 or placebo group developed long-term antitumour immunity after the AdB7-1/IL-2 treatment. These results are in line with our previous data showing that cured mice develop long-term immunity regardless of the cytokine vector used, although the different vectors exhibit higher or lower effectiveness in inducing complete tumour regression (To determine whether oestradiol supplement may affect the development of a tumour-specific CTL response, cured mice in both the E2 and placebo group were killed and their spleens removed 6 weeks following PyMT rechallenge. Splenocytes prepared from either group demonstrated a 50% specific lysis of 516MT3 cells, expressing the PyMT antigen, at an effector to target ratio of 90 : 1 (Figure 4In this study, we demonstrated that oestradiol, at physiologic concentrations, enhances the immune response by intratumoral adenoviral gene delivery of B7-1/IL-2 in a murine model of breast cancer in which the growth rate is increased by oestrogen. This difference was not due to an inability of oestrogen-depleted mice to induce a CTL response since cured mice from both groups exhibited similar CTL activity. However, we show that oestradiol increased the local IFN-γ response after delivery of AdB7-IL-2, which may be one mechanism to explain the higher response rate in the oestradiol-supplemented mice.γ indicating that treatment with Ad vectors induces cytokines and costimulatory molecules, which induce TH1-mediated antitumour responses (γ affects tumour cells to be more susceptible to T-cell-mediated killing (γ also appears to sensitise human breast cancer cells to undergo apoptosis (Distinct patterns of cytokine expression are associated with the development of TH1 or TH2 immune responses . Developγ production with no effect on IL-10 secretion (γ remained at the same levels seen with a low dose of oestradiol (γ after immune stimulation than those from males and an oestrogen-responsive element in the IFN-γ promoter in murine spleen cells has been found (It has been suggested that females produce stronger immune reactions than males (None of the larger established tumours underwent complete regression, but the tumours from oestradiol-treated mice showed a partial response and delayed tumour growth. This suggests that tumour size is a predictive factor for the success rate of immune gene therapy and that a dose adjustment or other adjuvant therapies are needed for treatment of heavy tumour burden. This is in line with current clinical principles where tumour size is a strong predictive factor for overall survival and relapse-free interval and larger tumours are treated with surgery in combination radiotherapy and/or chemotherapy (in vitro at low doses, it inhibits proliferation at high levels (In vivo, breast tumours may maintain high local levels of oestrogen independent on the overall circulating concentration suggesting that added exogenous oestrogen has very little effect on the local tumour environment (in vivo is dependent on the balance between all these effects. Moreover, oestrogen effects are related to the dose and type of hormone used. It is therefore important to use physiologic levels of oestrogen and the naturally occurring E2 in studies of pathogenesis and immune modulation.The results in our study are intriguing and somewhat unexpected since the cornerstone of treatment of oestrogen-dependent breast cancer is antioestrogen hormonal therapy. However, the action of oestrogen in breast cancer is complex, poorly understood and contradictory. While oestrogen stimulates tumour cell growth γ levels in tumour tissue, measured 2 days after tumour injection, which may be involved in the mechanisms leading to the higher response rate in these mice. Future studies will determine whether these data are valid for human breast cancer and how the partial oestrogen agonist tamoxifen, the most commonly used antioestrogen in breast cancer treatment, affects immune-stimulatory breast cancer therapies.We conclude that oestradiol increases the cure rate of oestrogen-enhanced murine breast cancer treated with an intratumoral adenoviral vector expressing B7-1/IL-2. Oestradiol treatment increased the IFN-


The longer survival of neoplastic patients achieved through improvements of therapeutic regimens has increased the relative risk of developing a second primary tumour (SPT). In this context, conventional cytopathology can define tumour histotype only in a small fraction of cases. In this study, we have evaluated whether selected combinations of monoclonal antibodies (MAbs) to tumour-associated antigens (TAAs) can increase the accuracy of conventional morphology in detecting second primary tumours (SPTs) in two particularly difficult areas of cytodiagnosis, namely that of effusions and pulmonary fine-needle aspirates (FNAs). The immunocytochemical (ICC) analysis of 334 cytological specimens demonstrated that the use of our selected panel of MAbs could allow a more efficient identification of SPTs in comparison with conventional morphology. This diagnostic improvement was statistically significant (P < 0.0001). The present findings show that the immunophenotyping of effusions and FNAs, providing a more accurate and objective identification of SPTs, may have significant therapeutic and epidemiological relevance.


A proteolysis-inducing factor (PIF) isolated from a cachexia-inducing murine tumour (MAC16) produced a decrease in body weight  within 24 h after i.v. administration to non-tumour-bearing mice. Weight loss was associated with significant decreases in the weight of the spleen and soleus and gastrocnemius muscles, with no effect on the weight of the heart or kidney and with an increase in weight of the liver. Protein degradation in isolated soleus muscle was significantly increased in mice bearing the MAC16 tumour. To define which proteolytic pathways contribute to this increase, soleus muscles from mice bearing the MAC16 tumour and non-tumour-bearing animals administered PIF were incubated under conditions that modify different proteolytic systems. In mice bearing the MAC16 tumour, there were increases in both cathepsin B and L, and the Ca2+-dependent lysosomal and ATP-dependent pathways were found to contribute to the increased proteolysis; whereas, in PIF-injected animals, there was activation only of the ATP-dependent pathway. Further studies in mice bearing the MAC16 tumour have provided evidence for increased levels of ubiquitin-conjugated proteins and increased mRNA levels for the 14 kDa ubiquitin carrier protein E2 and the C9 proteasome subunit in gastrocnemius muscle, suggesting activation of the ATP-ubiquitin-dependent proteolytic pathway. A monoclonal antibody to PIF attenuated the enhanced protein degradation in soleus muscle from mice bearing the MAC16 tumour, confirming that PIF is responsible for the loss of skeletal muscle in cachectic mice.