Tumour-bearing mice showed reduced carcass, muscle and fat mass compared with controls. EDL muscle performance and total daily activity were impaired in the tumour-bearing mice. Addition of single nutrients resulted in no or modest effects. However, supplementation of the diet with the all-in combination of high protein, leucine and fish oil significantly reduced loss of carcass, muscle and fat mass ) and improved muscle performance (loss of max force reduced to 55–64% of TB-con (P<0.05)). Moreover, total daily activity normalised after intervention with the specific nutritional combination (50% of the reduction in activity of TB-con (P<0.05)). In conclusion, a nutritional combination of high protein, leucine and fish oil reduced cachectic symptoms and improved functional performance in cancer cachectic mice. Comparison of the nutritional combination with its individual modules revealed additive effects of the single components provided.Cancer cachexia is characterised by metabolic alterations leading to loss of adipose tissue and lean body mass and directly compromises physical performance and the quality of life of cancer patients. In a murine cancer cachectic model, the effects of dietary supplementation with a specific combination of high protein, leucine and fish oil on weight loss, muscle function and physical activity were investigated. Male CD2F1 mice, 6–7 weeks old, were divided into body weight-matched groups: (1) control, (2) tumour-bearing, and (3) tumour-bearing receiving experimental diets. Tumours were induced by s.c. inoculation with murine colon adenocarcinoma (C26) cells. Food intake, body mass, tumour size and 24 h-activity were monitored. Then, 20 days after tumour/vehicle inoculation, the animals were killed and muscle function was tested  After acclimatisation for 1 week mice were divided into weight-matched groups: (1) control receiving control chow, (2) tumour-bearing receiving control chow, and (3) tumour-bearing receiving experimental diets. Data shown are derived from the combination of several experimental runs with identical animal characteristics and experimental procedures (unless stated otherwise) and differ only in the experimental diets used. All experimental procedures were approved by the Animal Ethical Committee  and complied with the principles of good laboratory animal care.Experiments are divided in: (A) experiments designed to test the effect of single or combined nutritional components (addition of high protein (hpr), leucine (leu), fish oil (fo)), added to the background diet (AIN93-M) and supplied as pellets; (B) experiments designed to test the effect of a complex nutritional combination that resembles the composition of the new generation FortiCare .The AIN93-M control diet in the category A experiments contained per kg feed: 126 g protein (100% casein), 727 g carbohydrates and 40 g fat (100% soy oil) . Experimental diets in this category were adjusted to control diets by partly replacing the carbohydrates and/or soy oil by protein and leucine , high protein and fish oil , or high protein and leucine and fish oil . The 22 g of fish oil contained 6.9 g EPA and 3.1 g DHA in a ratio of 2.2 : 1. In summary, in experiment A the following diets and interventions were compared: (1) no tumour, AIN93-M fed (Con); (2) tumour-inoculated AIN93-M fed (TB-con); (3) tumour-inoculated fed AIN93-M with additional protein and leucine (TB+hpr+leu); (4) tumour-inoculated fed AIN93-M with additional fish oil (TB+fo); (5) tumour-inoculated fed AIN93-M with additional fish oil and protein (TB+fo+hrp); (6) tumour-inoculated fed AIN93-M with additional fish oil, high protein and leucine (TB+fo+hpr+leu).In the category B experiment, the control diet was a more humanised diet, isocaloric and isonitrogenous to the control diet in the A category of experiments, and contained per kg feed 126 g protein (casein), 53 g fat (corn oil), and 699 g carbohydrates. The isocaloric experimental diet  contained per kg feed: 210 g protein (189 g intact protein of which 68% casein and 32% whey and 21 g free leucine), 53 g fat ), 561 g carbohydrates, 18 g galacto-oligosaccharides and 2 g fructo-oligosaccharides. In experiment B, diets were supplied as dough for product technical reasons.in vitro with RPMI-1640 supplemented with 5% fetal calf serum and 1% penicillin–streptomycin , tumour cells (5 × 105 cells in 0.2 ml) were inoculated subcutaneously into the right inguinal flank of the mice. Control (C) animals received a sham injection with 0.2 ml HBSS.Murine C-26 adenocarcinoma cells were cultured ex vivo in the category B experiment.Following inoculation of tumour cells or HBSS, body mass, food intake and tumour size (length and width) were measured three times a week. Only in the category B experiment, daily activity in the home cage was monitored. In all experiments, animals were anaesthetised and weighted at day 20 after tumour inoculation. Skeletal muscles , m. Gastrocnemius (mG), m. Extensor Digitorum Longus (mEDL) and M Soleus (mS)), the tumour, spleen, kidneys, liver, epididymal fat, thymus, lungs and heart were dissected and weighed. Carcass mass was calculated by subtracting tumour mass from body mass. In addition, muscle function was tested Physical activity was monitored continuously (24 h) during the 20-day study period starting at day 2, using activity sensors  that translated individual changes in the infrared pattern caused by movements of the animals into arbitrary activity counts. Sensors were mounted above the home cages and were connected through input ports and interface to a computer equipped with MED-PC IV software for data collection . Activity was expressed in counts per hour  and the light period (inactive period)). Activity was calculated for each mouse separately and was expressed relative to its own total activity on day 2, to correct for differences in the individual sensitivity of sensors. The activities of two subsequent days were averaged, to dampen the day to day variability. To determine changes in the activity pattern throughout the experiment, hourly and dark–light activity were expressed as percentage of total daily activity and translated into an actogram.ex vivo, as described previously . In experiment A different batches of animals were used, therefore, for all parameters it was defined that combination of data was allowed if no interaction between groups and experiments were present. Body composition data, tumour and organ masses on day 20 were compared between groups with analysis of variance (ANOVA) and Compared with control mice (Con), carcass and body weight were significantly lower in tumour-bearing control mice (TB-con) on day 20 after tumour inoculation . For allBody and carcass weight were significantly lower in tumour-bearing mice (TB-CON) compared with control mice (CON) on day 20 . The difOrgan (wet) mass of kidney, liver, thymus and heart either decreased with increased cachexia or showed no change . Nutritiex vivo in mEDL. Maximal force, maximal contraction velocity and maximal relaxation velocity were significantly different in TB-CON when compared with CON and TB-SNC  were determined d TB-SNC . When thd TB-SNC  and maxid TB-SNC . When mad TB-SNC  curve pod TB-SNC .P<0.01; RM-ANOVA) over the total period (2–19 days). Activity levels in TB-CON mice were significantly lower than in control mice on days 10–11 (P<0.05), and from day 16 onwards (P<0.01). The TB-SNC animals did not differ significantly from the control animals in their total activity throughout the experiment, whereas their activity was significantly higher at days 18–19 compared with TB-CON mice (P<0.05) (P<0.01), resulting in a drastic decrease in overall activity in the TB-CON mice. The TB-SNC mice were less active than control mice during the dark on day 18–19 (P<0.05), but more active than TB-CON mice on those days (P<0.05).Total daily activity showed a significant interaction between time and group ((P<0.05) . These d(P<0.05) . ThroughBesides a reduction in daily activity level of TB-CON mice, a clear shift in daily activity pattern was observed, that is, from dark to light, both in tumour-bearing controls and in TB-SNC animals on days 18–19 . To focuvs multiple components on body composition in the murine C26 carcinoma model was investigated. In contrast to supplementation of the single ingredients, the combination demonstrated a significant effect on the body composition. In addition, the specific nutritional combination also improved muscle function. Moreover, activity patterns as well as overall daily activity improved, probably as a consequence of improved body composition and muscle function. This clearly supports the added value of a multi target approach, in which catabolism (both protein and fat) is targeted through a reduction of inflammation through the addition of fish oil; whereas anabolism is specifically targeted by protein synthesis through the supplementation of high protein and leucine. The effects on body composition and performance are highly relevant to the clinical situation, because muscle function and daily activity are important contributors to the quality of life of the cancer patient  was the only group that showed significant differences  fat see ). The su fat see . These dOrgan (wet) mass of kidney, liver, intestine, thymus and heart were unaffected or decreased with increased cachexia. Nutritional supplementation resulting in increased carcass weight had no effect or partly normalised the loss in organ mass. Next to that, none of the selected ingredients increased tumour size. The complete nutritional combination showed a reduction in tumour size in experiment B.n=174), 1 year supplementation with BCAA resulted in a decreased prevalence of anorexia from 52 to 25%, whereas anorexia prevalence remained unchanged in the control group -deficient rats (Further studies in patients are needed to confirm the anti-cachectic properties of a nutritional supplement containing at least leucine-enriched high protein and fish oil. Also the mechanistic effects of the nutritional combination on inflammatory processes warrant further research. When anorexia is present in this patient group, the use of a high caloric supplement seems valid. In such a setup, the effect of BCAAs in the nutritional combination on both anorexia and cachectic parameters could be evaluated. Moreover, based on the results from this study it is clear that more attention should be paid to prevention of cachexia to maintain quality of life for the patient.In conclusion, this specific nutritional combination of high protein, leucine and fish oil improved the cachectic outcome of mice inoculated with the C26 adenocarcinoma cell line. The carcass, fat and muscle mass increased and the muscle function and daily activity improved when compared with tumour-bearing mice on the control diet. These data show that single ingredient interventions have limited value, and support the need for a balanced combination of different ingredients to enable a multi-targeted intervention to achieve beneficial effects in the complex conditions of cancer cachexia, as reflected in changes of body composition, muscle functionality and daily activity. The findings are clinically highly relevant, because muscle function and daily activity contribute to a great extent to the quality of life of the cancer patient (


To determine the effects of 211At-labelled antibodies in solid tumour tissue, nude mice carrying OHS human osteosarcoma xenografts received intratumour injections at dosages of 1, 2 or 4 MBq (-1) tumour. The radioisotope was conjugated to either the osteosarcoma-specific monoclonal antibody TP-3 or the non-specific polyclonal antibody hlgGkappa. Tumour retention of injected radioimmunoconjugate (RIC), measured as the percentage of injected activity dosage per gram, was significantly higher for the [211At]TP-3 (203 +/- 93 at 24.1 h post injection) compared with the [211At]hlgGkappa (57 +/- 22 at 23.2 h post injection). The radioactive count rates in body (measured at neck and abdomen) were significantly lower with the TP-3 than with the hlgGkappa. Microautoradiography of the tumour radionuclide distribution was different for the two RICs, i.e. the [211At]TP-3 was to a larger extent concentrated near the injection site, whereas the [211At]hlgGkappa was more evenly distributed all over the tumour. The tumour growth was significantly delayed as a function of the injected activity dosage but without significant difference between the specific and the non-specific RIC. According to this study, it is possible to deliver highly selective radiation doses to solid tumours using intratumour injection of alpha-particle-emitting RICs. Improved tumour retention caused by antigen binding indicates that reduced normal tissue exposure can be obtained with antigen-specific antibodies. The heterogeneous tumour dose distribution observed is, however, a major impediment to the use of alpha-particle emitters against solid tumours.


A series of monomeric porphyrins (2-8) based on porphyrin C (1) have been tested as sensitisers for photodynamic therapy (PDT) of cerebral glioma using the in vitro/in vivo C6 intracerebral animal tumour model. The in vivo screening, consisting of cytotoxicity, phototoxicity (red light) and subcellular localisation studies, revealed two sensitisers , which had greater photoactivity than porphyrin C and similar photoactivity to haematoporphyrin derivative (HpD) although at a 5-fold higher dose than HpD. Both sensitisers showed intracellular localisation to discrete organelle sites and exhibited considerably less 'dark' cytotoxicity than HpD. The kinetics of uptake of porphyrins 7 and 8 was studied in the mouse C6 glioma model as well as in biopsy samples from normal brain, liver, spleen and blood. Maximal drug uptake levels in tumour occurred 9 and 6 h after intraperitoneal injection for 7 and 8 respectively, at which time the tumour to normal brain ratios were 15:1 and 13:1 respectively. The effect of PDT using porphyrin 7 activated by the gold metal vapour laser tuned to 627.8 nm was studied in Wistar rats bearing intracerebral C6 glioma. At a drug dose of 10 mg porphyrin 7 kg-1 body weight and laser doses of up to 400 J cm-2 light, selective tumour kill with sparing of normal brain was achieved, with a maximal depth of tumour kill of 1.77+/-0.40. mm. Irradiation following a higher drug dose of 75 mg porphyrin 7 kg-1 body weight resulted in a greater depth of tumour kill, but also significantly increased the likelihood and extent of necrosis in normal brain.


When combined with radiation or chemotherapy, AQ4N substantially increased the effectiveness of these modalities in a range of in vivo model systems. AQ4N potentiates the action of radiation in both a drug and radiation dose-dependent manner. Further the enhancement observed is schedule-independent with AQ4N giving similar effects when given at any time within 16 h before or after the radiation treatment. In combination with chemotherapy it is shown that AQ4N potentiates the activity of cyclophosphamide, cisplatin and thiotepa. Both the chemotherapeutic drugs and AQ4N are given at doses which individually are close to their estimated maximum tolerated dose (data not included) which provides indirect evidence that in the combination chemotherapy experiments there is some tumour selectivity in the enhanced action of the drugs. © 2000 Cancer Research CampaignAQ4 ethyl]amino}5,8-dihydroxyanthracene-9, 10-dione) is a prodrug designed to be excluded from cell nuclei until bioreduced in hypoxic cells to AQ4, a DNA intercalator and topoisomerase II poison. Thus, AQ4N is a highly selective bioreductive drug that is activated in, and is preferentially toxic to, hypoxic cells in tumours. Five murine tumours  have been used to investigate the anti-tumour effects of AQ4N. In only one tumour (MAC16) was AQ4N shown to be active as a single agent. However, when combined with methods to increase the hypoxic tumour fraction in RIF-1  and MAC26 tumours  there was a substantial enhancement in anti-tumour effect. Notably, RIF-1 tumours treated with AQ4N (250 mg kg


The interstitial fluid pressure (IFP) and the proton spin-lattice and spin-spin relaxation times (T1 and T2) of some experimental tumours have been shown to be related to tumour water content. These observations have led to the hypothesis that magnetic resonance imaging (MRI) might be a clinically useful non-invasive method for assessment of tumour IFP. The purpose of the work reported here was to examine the general validity of this hypothesis. R-18 human melanoma xenografts grown intradermally in Balb/c nu/nu mice were used as the tumour model system. Median T1 and T2 were determined by spin-echo MRI using a 1.5-T clinical whole-body tomograph. IFP was measured using the wick-in-needle technique. No correlation was found between tumour IFP and fractional tumour water content. Moreover, there was no correlation between median T1 or T2 and IFP, suggesting that proton T1 and T2 values determined by MRI cannot be used clinically to assess tumour IFP and thereby to predict the uptake of macromolecular therapeutic agents.


A human malignant pleural mesothelioma was xenografted serially in athymic nude Rowett rats for 27 passages during 33 months. After the two initial passages (P), the take rate during P3-9 was 100% (192/192). The tumour grew progressively during P3-9 in 99% (190/192) and regressed totally in 1% (2/192). The take rate for the tumour xenografted to athymic BALB/c mice was also 100% (17/17) and no regressions were observed. During serial passaging in nude rats, the tumour-volume doubling time (TD) decreased from 6 days in P2 to 3 days in P8-9 (P less than 0.001) and then remained around 3 days during P10-25. A TD of 11 days in P1 (man-mouse) for tumours grown in mice decreased during 10 passages in rats to 4 days (P less than 0.005) when the tumour was transplanted to mice in P11. Light microscopic morphology of the tumour was retained in rats and mice. We believe that our experimental tumour model using the nude rat as a carrier of the xenograft will be useful for studies of human mesothelioma.


Recent data on extrapulmonary colony formation after heparin administration are inconclusive. A systematic study of this topic was undertaken with 4 experimental tumour systems and 2 distinct periods of reduced clotting capacity in rats and mice. I.v. injection of various numbers of tumour cells into i.p. heparinized animals leads to: (1) Significant reduction in the number of lung colonies. The effect after 9 h anti-coagulation is equal to or probably greater than after 2 h. (2) The reduction in the number of lung colonies caused by heparin is independent of the number of cells injected. (3) The number of extrapulmonary extrathoracic colonies is significantly increased by heparin in 3 of the 4 tumour systems. (4) The number of extrapulmonary intrathoracic colonies is probably unaffected. (5) The increase in extrapulmonary extrathoracic colony formation is not related to the degree of reduction in lung colonies. These data lead to the conclusion that the capacity of the lung capillaries to trap tumour cells can be decreased by heparin-induced alterations in fibrin formation. This results in a lodgement of tumour cells throughout the body which is far more pronounced than in animals with normal clotting capacity.


We assessed the prognostic significance of tumour infiltrating lymphocytes (TIL) in intestinal-type gastric cardiac cancer.Recent evidence suggests that CD4Tumour infiltrating lymphocyte (TIL) subsets and tumour infiltrating macrophages (TIM) were investigated in 52 cases using tissue microarrays. The interrelationship between the cell populations  in different compartments and NED-survival was investigated (median follow-up time: 61 months).+ Treg in the tumour stroma (>125.9 FoxP3+TILs/mm2) had a median survival time of 58 months while those with low FoxP3+ TIL counts (<125.9 FoxP3+TILs/mm2) had a median survival time of 32 months (p = 0.006). Patients with high versus low stromal CD68+/FoxP3+ cell ratios in primary tumour displayed median survivals of 32 and 55 months, respectively (p = 0.008).Intraepithelial infiltration with TIL and TIM including Treg was generally low and not related to NED-survival. However, patients with large numbers of FoxP3Our results suggest that inflammatory processes within the tumour stroma of gastric intestinal-type adenocarcinomas located at the gastric cardia may affect outcome in two ways. Tumour-infiltrating macrophages are likely to promote carcinogenesis while large numbers of Treg are associated with improved outcome probably by inhibiting local inflammatory processes promoting carcinogenesis. Thus, inhibition of Treg may not be a feasible treatment option in gastric adenocarcinoma. Gastric carcinoma is the third most common gastrointestinal tumour type in developed countries . Similar+CD25+FoxP3+ regulatory T-cells (Treg) are able to induce tolerance to self-antigens and may also inhibit anti-tumour immune response. Tregs can inhibit effector T-cells, suppress the antigen presenting function and promote the immunosuppressive function of dendritic cells, monocytes and macrophages [Escape from immunosurveillance is a fundamental step in cancer development. There is accumulating evidence indicating that CD4rophages .+ and CD8+ TIL were associated with improved survival [+ and cytotoxic TIL were prognostically favourable, while Treg presence had no influence on prognosis [The prognostic significance of tumour infiltrating lymphocytes (TIL) in cancer therapy is not fully understood. A beneficial prognostic effect of increased lymphocyte infiltration has been shown for various tumour types. In ovarian carcinoma both intratumoural CD3survival ,8 while survival . An inverognosis . A recenrognosis .We investigated the prognostic value of infiltration by various TIL-subgroups and macrophages including Treg in adenocarcinoma of the cardia in both primary tumour and draining lymph nodes.Between 1993 and 2004, 135 patients with gastric adenocarcinoma of the cardia underwent evaluation and primary surgical treatment at the Erlangen University Hospital. A subgroup of 52 patients was enrolled in the present study according to the following criteria: All patients were required to have invasive gastric adenocarcinoma of the intestinal subtype according to the Laurén classification without distant metastases at presentation. Patients with oesophageal Barrett's adenocarcinoma were excluded. An exclusively surgical treatment regimen with complete resection (R0) was performed. No patient received (neo)adjuvant radiotherapy or chemotherapy. Staging was recorded according to the International Union against Cancer system (UICC 2002). We gathered data concerning recurrence and survival both from our university hospital, the patients' physicians and the respective residents registration office. The minimum follow-up time was 36 months; the mean follow-up time was 71.2 months (median 61.0 months) with a 95% confidence interval of 54.7 to 57.8 months. The use of the tissue sections and the patients' survival data following patient consent was approved by the Friedrich-Alexander University Erlangen-Nuremberg ethics committee.474 core biopsies from primary tumour, uninvolved draining lymph nodes and, if applicable, metastatic nodes were processed into tissue microarrays (TMA) Figure . As guid2, mean epithelial surface area per case: 0.241 mm2, mean stromal surface area per case: 0.225 mm2). In stromal tissue, only areas adjacent to tumour infiltration were selected. In uninvolved lymph-nodes, two images were acquired per core adding to a total of six images per case.Evaluation of the immunostained microarrays was performed using a standard light microscope at a magnification of ×200, a CCD-camera and a semi-automatic image processing software . Labelled lymphocytes and macrophages were counted. In each image surface areas of the epithelial and the stromal compartments were calculated and glandular luminal areas were subtracted. Thus, it was possible to assess separately intraepithelial and stromal lymphocyte and macrophage infiltration both in the primary tumour site and lymphatic metastatic tissue Figure . For eacClinical variables evaluated were age, gender, UICC stage, T category and N category. Overall survival was defined as the period from surgery until death or end of follow-up. No-evidence-of-disease (NED) survival was evaluated as the period from surgery until evidence of recurrence . A univariate regression analysis of NED-survival was performed using Cox's proportional hazards model . For a multivariate analysis of NED-survival we adopted only variables being of significant impact in univariate analysis. Survival was analyzed by the Kaplan-Meier method . The logThe patients' clinical and histological characteristics are depicted in Tables Due to the clear border between the stromal and epithelial compartment in intestinal-type carcinomas, we evaluated the two compartments separately. A pronounced difference in TIL/TIM- infiltration between these stromal compartments was detected both at primary tumour site and in lymph node metastasis Table .All TIL subsets and macrophages were found predominantly in stroma adjacent to epithelial tumour cell clusters while they occurred only infrequently between epithelial cells within the tumour cell nests Figures  and 2.The proportion of Tregs of total T cell counts was larger in areas of tumour infiltration  than in the control lymph nodes Table .+ TIL infiltration of the tumour stroma at the primary site was of pronounced prognostic significance: patients with high stromal FoxP3+ TIL counts (>125.9 FoxP+ TIL/mm2) had a median survival time of 58 months while those with low FoxP3+ counts (<125.9 FoxP3+ TIL/mm2) had a median survival time of 32 months  at the primary tumour site had a median survival of 32 months while those with a lower CD68+/FoxP3+ cell ratio (< 2.9) had a median survival of 55 months (p = 0.008) (graph not shown). Considering the uninvolved regional lymph nodes alone, higher numbers of Granzyme B+ cells were linked to a beneficial outcome .The ratio of stromal CD8ce Table . In surv+ TIL infiltration was inversely correlated with UICC-stage , number of lymph node metastases  and N category in general . For other cell types, no such correlation could be found.Early disease (UICC stage I) was associated with significantly better NED-survival rates compared to advanced disease (UICC II-IV) . An increasing stromal FoxP3+ Treg in colorectal cancer also showed high Treg numbers to be associated with improved survival [Tregs inhibit the function of effector cells ,6. Consesurvival .+CD25+ TIL as compared to control mesenteric nodes. However, no specific correlation between survival and intensity of Treg infiltration could be demonstrated [Studies on gastric carcinomas have also yielded conflicting results. Kono et al. found a higher percentage of Treg among PBMCs in patients with advanced gastric carcinoma. Higher numbers of Treg in PBMCs had an adverse effect on outcome, and numbers of Treg declined to normal values following curative resection . Mizukamnstrated . These pFor these reasons, we have decided to focus on a morphologically homogeneous group of intestinal-type adenocarcinomas located at the gastric cardia. We have assessed the numbers of tumour-infiltrating immune cells in different tumour-related compartments in relation to outcome parameters. We show firstly that the presence of various TIL subsets including Treg, cytotoxic T-cells as well as tumour-infiltrating macrophages within the epithelial compartment had no significant influence on survival rates. This is in contrast to our previous analysis of anal carcinomas . This maOn the face of it, these findings are unexpected and contrast previous studies of ovarian carcinoma -9,19. NeChronic inflammation, either related to H. pylori infection or to autoimmune gastritis, is considered the major risk factor for the development of gastric cancer of intestinal type. This is probably also true for gastric adenocarcinoma located at the cardia . DevelopWe also find that a higher number of Granzyme B positive T cells in uninvolved lymph nodes is associated with a better outcome. Granzyme B is a key cytolytic mediator secreted by activated cytotoxic T cells. A large systemic number of activated cytotoxic T cells as represented by large numbers in uninvolved lymph nodes may be an indicator of an efficient systemic host anti-tumor immunity .Moreover, we show that large numbers of tumour-associated macrophages in conjunction with small numbers of Treg are associated with poor prognosis. This is in agreement with a previous study . It is nOur results suggest that inflammatory processes within the tumour stroma of intestinal-type adenocarcinoma of the gastric cardia may affect outcome in two ways. Tumour-infiltrating macrophages are likely to promote carcinogenesis as has been reported previously for various tumour models. By contrast, Treg may down-modulate local inflammatory processes which promote carcinogenesis. Thus, inhibition of Treg may not be a feasible treatment option in gastric adenocarcinoma.The authors declare that they have no competing interests.MH: conception and design, analysis and interpretation of data, drafting of manuscript. AD: analysis and interpretation of data. WH: conception and design. GGG: conception and design, revision of manuscript. GN: conception and design, revision of manuscript. LVD: conception and design, interpretation of data, drafting of manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/9/65/prepub


Tissue histamine levels were studied in C3H and C57BL/6 mice bearing a methylcholanthrene-induced fibrosarcoma, in Wag rats bearing an aflatoxin B1-induced hepatoma, and in Commentry rats bearing a grafted hepatoma. Histamine levels were significantly higher (1.5 to 3 fold) in the tumour-bearing animals for ventral and dorsal skin, skeletal muscle and stomach fundus. Total histamine content was increased in the spleen. In C3H mice with McC3-1 fibrosarcoma, the excision of the tumour or its partial regression by intratumoral injections of corynebacterium parvum induced a reversion to normal values. The tumour thus appears responsible for the increased histamine levels in tissues distant from the tumour.


The anti-tumour activity induced by glucans  and fructosans (levan and carboxymethyl-levan) was compared with the activity of C. parvum. The following effects on tumour systems in CBA mice were assayed: (a) adjuvant activity on the immune response against tumour-specific transplantation antigens (TSTA) with a methylcholanthrene-induced fibrosarcoma; (b) cytostatic activity of peritoneal macrophages against radiation-induced leukaemia cells; and (c) inhibition of tumour nodule formation in the lungs following i.v. injection of fibrosarcoma cells. All the polysaccharides induced cytostatic macrophages, but the dextrans and levans did so only after i.p. and not i.v. injection. Only lentinan, yeast cell walls and pseudonigeran were active in the lung-nodule inhibition test; and only lentinan and dextran sulphate showed slight adjuvant activity for TSTA. It is concluded that the anti-tumour activity induced by these polysaccharides is predominantly non-specific macrophage-mediated and much weaker than that found with C. parvum.


Tumour volume was monitored for 90 days or until a target volume (3.5 ml) was reached. No differences were seen between the first three groups, with all tumours reaching the target volume by 8–11 days. A total of 13 and 15% of tumours did not reach the target volume by day 90 following HT or ALA-PDT treatment, respectively. ALA-PDT+HT showed the greatest antitumour effect (P=0.0001), with 61% of the tumours not reaching the target volume. Viability and in vitro growth were also assessed in cells from tumours excised after treatment. ALA-PDT+HT reduced the fraction of viable tumour cells by 85%, and in vitro culture showed pronounced growth delay compared to control cells. These results demonstrate an enhanced antitumour effect upon ALA+HT, which appears to involve direct cell toxicity rather than solely vascular damage.The possibility of enhancing aminolaevulinic acid (ALA)-based photodynamic therapy (PDT) by simultaneous application of localised hyperthermia (HT) was evaluated. Treatments of rat DS-sarcomas included: (i) control, (ii) ALA administration (375 mg kg One approach uses a natural porphyrin precursor, 5-aminolaevulinic acid (ALA), which enters the endogenous haem synthesis pathway. The final step in this pathway involves the incorporation of iron into protoporphyrin IX (PpIX) to produce haem. Due to the fact that many tumours exhibit abnormal levels of some of the enzymes involved in this pathway, a selective accumulation of PpIX – which is an efficient photosensitiser – can occur in tumour cells  was used in all experiments. Solid tumours grew subcutaneously following injection of DS-sarcoma ascites cells into the hind foot dorsum . This tumour was originally chemically induced in rats and arose in the auditory canal . After ALA injection, the animals were kept under subdued lighting conditions until treatment commenced. An equivalent volume of PBS was administered to control animals. The ALA dose was chosen on the basis of preliminary experiments, showing this dose to be the most effective in terms of tumour growth inhibition in combination with low toxicity. These experiments also showed this dose to be the lowest possible ALA concentration resulting in maximum ALA-induced fluorescence in the tumour investigated.5-Aminolaevulinic acid  was dissolved in phosphate-buffered saline (PBS) at a concentration of 80 mg ml−1 i.p., Narcoren®, Merial, Germany) and randomly allocated to the various experimental groups, the details of which are summarised in Table 1Once a tumour volume of 0.6–0.8 ml had been attained (6–8 days after tumour implantation), the animals were anaesthetised with sodium pentobarbital  and the tumours heated at a rate of 0.4°C min−1 to a set temperature of 43°C (tumour centre) using the radiation source. Following a 20 min heat-up phase, the tumour temperature was maintained at this set level for 60 min, by continuous regulation of the source that switched on and off intermittently . This resulted in an average effective total energy dose of 1120 J cm−2 over the treatment period as measured in pilot studies. These pilot studies, in which HT was applied were also used to obtain information concerning the pattern of irradiation with the light delivery system used. An evaluation of these studies showed that, on average, tumours of the volume used in the present study were irradiated for a period of 1.3 s followed by a nonillumination period of 2 s, with this pattern continuing over the 80 min illumination period. This pattern was subsequently used for the groups requiring ‘nonthermal’ illumination so that the light delivered in the range from 420 to 800 nm was equal for all the illuminated groups, with a fluence rate of 200 mW cm−2 over this wavelength range for all treated groups. Additionally, the light hyperfractionation schedule was the same for all groups receiving irradiation. Light dosimetry was performed using a calibrated radiometer/photometer . Irradiation was applied solely to the tumour, the remainder of the animal being shielded by aluminium foil.A radiation delivery system was used which has been described in detail previously . The ligOn considering the treatment data outlined in d) of the tumours were measured on a daily basis and the tumour volume (V) was determined using the ellipsoid approximation V=π/6 × d1 × d2 × d3. In accordance with the guidelines of the UKCCCR  using a glass pestle. A single-cell suspension was then prepared by incubating the tissue clumps in 30 ml culture medium (RPMI1640 (Sigma), supplemented with 10% bovine calf serum ) with 2 ml of an enzyme mixture containing 0.4 mg ml−1 DNAase  and 0.05 mg ml−1 collagenase (Sigma) for 20 min at room temperature, after which the cells were dispersed by repeated passage through a 22 gauge needle. Finally, the cells were washed, centrifuged and resuspended in culture medium . The cell number was then assessed in a haemocytometer, and cell viability determined by trypan blue exclusion . The cells were then decanted into tissue culture flasks at an initial concentration of 50 000 vital cells per millilitre growth medium, where they grew as a suspension in a humidified atmosphere of 5% CO2 in air for up to 6 days in the dark. The number of vital tumour cells was re-evaluated on a daily basis in a haemocytometer.In a second series of experiments, tumours were treated In order to assess differences in PpIX accumulation between tumour tissue and normal tissues, tumour-bearing animals were injected with ALA or PBS as described above. At 3 h after injection, tumours and normal tissues  were excised and mechanically dispersed to obtain a single-cell suspension. Flow cytometric measurements were performed using an EPICS XL-MCL system  to determine ALA-induced fluorescence. For this, the cells were illuminated by an argon laser (wavelength 488 nm) and the mean fluorescence was assessed in the wavelength range 605–635 nm. Tissues of animals receiving PBS served as controls and ratios between ALA-treated and control tissues were calculated.In vivo tumour growth characteristics were calculated using Kaplan–Meier statistics and the significance of the differences between the various groups was assessed using a log-rank analysis. All other analyses were carried out using the Wilcoxon test for unpaired samples. The significance level in all cases was set at α=5%.P=0.0001, in both cases). Although the probability of not reaching the tumour target volume 90 days after treatment was approx. 15% in the case of either ALA-PDT or HT treatment, the log-rank test showed the course of the curves from these two treatment groups to be significantly different (P=0.0006), with the ALA-PDT group demonstrating an enhanced tumour growth delay up to 30 days following treatment, as seen in the corresponding right-shift in the early phase of the Kaplan–Meier curve. Treatment of tumours with ALA-PDT+HT produced the most pronounced antitumour effect, with a probability of the tumour not reaching 3.5 ml by 90 days after treatment of 61%, an effect which was significantly greater than that seen in the control, HT and ALA-PDT treatment groups . Of the animals monitored till 90 days in the ALA-PDT+HT group, none showed any evidence of tumour presence on macroscopic examination. The combined treatment therefore produces an antitumour effect which appears to be more than additive in comparison to the two component treatments alone. It is important to note that the assessment of true synergism, as described by n=25) prior to the treatment to 37.6±0.16°C (175 temperature measurements) during the treatment, with a peak temperature of 38.4±0.12 (n=25). These temperatures are within the normal physiological range for rat rectal temperatures and therefore an HT component in the ALA-PDT treatment is unlikely. Experiments comparing the effects of fractionated and continuous light delivery schedules in ALA-PDT treatment showed no significant difference on log-rank analysis, with the probability of the tumour not reaching the target volume of 14.5% (n=25) by 90 days with the fractionated light schedule and 15% (n=11) with the continuous light schedule. Thus an influence of light fractionation on the PDT effect in this setting can be ruled out.Following treatment, animals from all groups were found to recover rapidly, with no side effects on general well-being or phototoxic effects on normal skin being recorded. Tumour size was assessed either until a tumour volume of 3.5 ml was attained  or up until 90 days after treatment. The probability of the tumour volume being less than 3.5 ml at any time point during the study was calculated using a Kaplan–Meier analysis . Subsequently, the growth of viable cells from tumours of the different treatment groups was monitored in vitro .In further experiments in which tumours were excised immediately after treatment, the degree of cell viability was assessed , simultaneous PDT and HT could not be carried out, even though this may be desirable when considering application in the clinical setting , which was considerably greater than that achieved in a mouse colon carcinoma model with a lower temperature of 40.5°C, where only a growth delay was seen (ne cells . A similIn conclusion, a tumour therapy is described, which is based on ALA-PDT being carried out simultaneously with 43°C HT using a single, easily applied, nonlaser radiation source. This combined treatment was found to be more effective than the sum of its components, an effect which appears not to be based solely on vascular damage. Investigations into the mechanisms underlying the effects of ALA-PDT+HT are to be carried out to more clearly define the interaction of the therapy components.


Tumours ferment glucose to lactate even in the presence of oxygen . The pentose phosphate pathway (PPP) allows glucose conversion to ribose for nucleic acid synthesis and glucose degradation to lactate. The nonoxidative part of the PPP is controlled by transketolase enzyme reactions. We have detected upregulation of a mutated transketolase transcript (TKTL1) in human malignancies, whereas transketolase (TKT) and transketolase-like-2 (TKTL2) transcripts were not upregulated. Strong TKTL1 protein expression was correlated to invasive colon and urothelial tumours and to poor patients outcome. TKTL1 encodes a transketolase with unusual enzymatic properties, which are likely to be caused by the internal deletion of conserved residues. We propose that TKTL1 upregulation in tumours leads to enhanced, oxygen-independent glucose usage and a lactate-based matrix degradation. As inhibition of transketolase enzyme reactions suppresses tumour growth and metastasis, TKTL1 could be the relevant target for novel anti-transketolase cancer therapies. We suggest an individualised cancer therapy based on the determination of metabolic changes in tumours that might enable the targeted inhibition of invasion and metastasis. Cancer is now viewed as a disease resulting from cancer-causing genes that deregulate cellular proliferation, differentiation, and death. Genetic alterations acquired by tumours also modify their biochemical pathways, resulting in abnormal metabolism. Warburg proposed a model of tumorigenesis involving altered energy production in tumours. He identified a particular metabolic pathway in carcinomas characterised by the anaerobic degradation of glucose even in the presence of oxygen (aerobic glycolysis) that leads to the production of large amounts of lactate .in vitro, and also to inhibit cell proliferation or suppress tumour growth in mouse models or cancer patients as a result of reduction of transketolase activity (The nonoxidative part of the PPP is controlled by thiamine- (vitamin B1) dependent transketolase enzyme reactions. Transketolase enzyme reactions of the nonoxidative part of the PPP enable oxygen-independent glucose degradation, and play a crucial role in nucleic acid ribose synthesis utilising glucose carbons in tumour cells. More than 85% of ribose recovered from nucleic acids of certain tumour cells is generated directly or indirectly from the nonoxidative pathway of the PPP  has been performed. This approach resulted in the identification of two novel small-molecule inhibitors, which inhibit human TKT and suppress proliferation of cancer cell lines , and transketolase-like-2 (TKTL2) to tumour-specific transketolase metabolism have not been investigated. Here, we provide evidence that TKTL1 mRNA and protein are specifically overexpressed in tumours, whereas TKT and TKTL2 expression are not upregulated. We demonstrate that TKTL1 protein is expressed in invasive tumours and predicts poor patient survival in colon and urothelial cancer. Our findings strongly indicate that overexpression of TKTL1 is responsible for the observed tumour-specific effects of transketolase enzyme reactions, and represents the basis for the observed inhibition of proliferation of cancer cells by anti-transketolase approaches.Real-time PCR-based transcript quantification and Western blot analysis have been described previously  with colon adenocarcinoma were included. All patients underwent colectomy, and 30% of colon adenocarcinomas were diagnosed as noninvasive (pTis), whereas 70% of colon adenocarcinomas were invasive at the time of diagnosis. Eleven tumours were classified as pT1, eight as pT2, 14 as pT3, and 16 as pT4, according to the UICC classification.in situ) and 31 were classified as muscle-invasive (⩾pT2). Overall, 42% of the tumours showed no or only weak staining (staining score 0 or 1), 10% showed some staining (score 2), and 48% showed strong staining (score 3).A total of 64 patients (median age 67.5 years) with urothelial carcinoma were enrolled in this study. Of these, 59 patients underwent treatment for urothelial carcinoma in which: 21 underwent transurethral resection for superficial bladder cancer; 22 underwent radical cystectomy; 16 underwent surgery for upper urinary tract carcinomas; and five underwent surgery for benign reasons. At the time of surgery, 14 % (8 out of 59) of the patients had lymph node metastases. Overall, 28 tumours were classified as nonmuscle-invasive , and the University of Palermo, Department of Surgical and Oncological Science, Surgery Pathophysiology Section. None of the patients received neoadjuvant radiotherapy or chemotherapy. From each patient, cancerous and normal tissue was available. For RNA extraction, the specimens taken during the operation were immediately snap-frozen in liquid nitrogen and subsequently stored at −80°C until use. For immunohistochemistry (IHC), the specimens were fixed in 3.4% buffered formalin for 24 h and embedded in paraffin. Histological diagnosis was performed by three independent, experienced pathologists .μm thick paraffin sections were analysed by IHC. Dewaxed sections were heated for antigen unmasking in 10 mM sodium citrate (pH 6.0) in a microwave oven for 1 min at 450 W followed by 5 min at 100 W. After rinsing in dH2O, inhibition of endogenous peroxidase was performed with a 5 min incubation with 3% H2O2. Endogenous avidin-biotin was blocked by the use of a commercial biotin blocking system (DAKO) for 10 min. After two washes in Tris/saline buffer (TBS), slides were incubated with 1% goat serum for 30 min to block unspecific staining. Sections were subsequently exposed to mouse anti-TKTL1  antibody (15 μg ml−1) or anti-Ser473 phospho-Akt  overnight at 4°C. The monoclonal anti-TKTL1 antibody JFC12T10 has been described previously  and diaminobenzidine tetrahydrochloride (DAB) , and counter-stained with Mayer's haematoxylin .For scoring of TKTL1 expression, a scale from 0 to 3 was defined as: score 0 indicates 0–20%, score 1 indicates 21–50%, score 2 indicates 51–80%, and score 3 indicates >80% of the tumour cells were stained for TKTL1.Identification of genes selectively expressed or overexpressed in tumours is a crucial prerequisite for molecular diagnosis and treatment of cancer by addressing molecular targets. To identify such targets, we used the real-time PCR technique. When comparing the transcript levels from five colon cancer tissues to nontumour samples from the same patients, we initially detected a 35-fold overexpression of TKTL1 in one colon carcinoma sample. As TKTL1 is one of three highly similar transketolases encoded by three separate genes  that specifically detects TKTL1  as well In some cases of undifferentiated gastric carcinoma, strong nuclear expression was observed . Analysiβ-oxidation of fatty acids  with colon adenocarcinoma was performed. Non-neoplastic colon tissues examined did not reveal TKTL1 expression, and noninvasive colon cancer specimens were negative or barely positive for TKTL1 staining . In contP=0.001), and is shown in the Kaplan–Meier plot  3% had score 0, 3% had score 1, 13% had score 2, and 81% had score 3 TKTL1 staining. There was statistically significant correlation between staging and staining intensity . The overall disease-specific 5-year survival rate was 47% . Patients dying because of tumour progression showed in 83% most intensive staining pattern of the tumours. Three patients were lost to follow-up and four patients died because of other reasons. Of 10 tumours initially found to be metastatic, nine showed strong overexpression of TKTL1 (grade 3). The correlation between staining intensity and patient survival was significant . Furthermore, it has been shown that both glucose usage and lactate production represent markers indicating poor prognosis (2CO3 if CO2 is produced by the oxidative part of the PPP. Both lactate acid and H2CO3 production lead to excretion of protons and a decline in pH of the surrounding matrix. This tissue acidosis triggers a p53-mediated cell death of neighbouring healthy cells (The PPP allows nonoxidative glucose degradation and enables a lactate-based matrix-degradation of the surrounding tissue (hy cells .Although aerobic glycolysis leads to the above-mentioned selective growth advantages, the most important question remains unresolved. What is the energetic basis of anaerobic glucose degradation? An analysis of the contribution of different fuels and metabolic pathways in proliferating MCF7 breast cancer cells has demonstrated that 65% of the total ATP turnover is from unidentified sources (While considering the above-listed advantages the nonoxidative PPP confers upon tumour cells, it is important to stress that the PPP is energetically as inefficient as the anaerobic glucose degradation via the Embden-Meyerhof pathway. When oxygen is absent, glucose degradation via both the Embden-Meyerhof pathway and the PPP leads to lactate production. Muscles perform the final mitochondrial oxidative steps of oxidative phosphorylation only if oxygen is present (Pasteur effect; 14C isotope labelling and its distribution in carbon atoms of fructose-6-phosphate differs from that predicted by reaction sequences (The energetic output of anaerobic glucose degradation, either based on the Embden-Meyerhof pathway or the PPP reactions as presented in text books, is worse than the energetic output of oxidative glucose degradation. Interestingly, the proposed reactions of the nonoxidative PPP are not firmly established; experimentally measured degree of de novo fatty acid synthesis, inhibition of Embden-Meyerhof glycolysis, enhanced lactate production, and mitochondria-independent ATP generation. Such postulated metabolic changes indeed have been observed in tumours. Enhanced glucose usage (de novo fatty acid synthesis upregulation (During the evolution of higher vertebrates, mutations in the TKTL1 gene have led to an altered substrate specificity and altered enzymatic reaction kinetics. An enhanced, one-substrate reaction allowing the use of xylulose-5-phosphate as sole substrate has been detected (gulation , pyruvatAs lactate and pyruvate regulate hypoxia-inducible gene expression independently of hypoxia by stimulating the accumulation of hypoxia-inducible Factor 1 alpha (β-oxidation of fatty acids. Recent studies have shown that this metabolic adaptation for an efficient growth is present in tumours. In tumours, activated Akt inhibits β-oxidation of fatty acids (β-oxidation of fatty acids.The presence of the TKTL1 pathway in tumor cells would also explain why tumor cells with an inhibited mitochondrial respiration and a concomitant enhanced aerobic glycolysis are less sensitive to induction of apoptosis by common anticancer agents, but highly sensitive to an inhibition of aerobic glycolysis (β-oxidation by activation of Akt or inhibition of AMPK. Future TKTL1-based anticancer therapy may also be improved by substrate limitation through application of a ketogenic diet (All the observed metabolic changes in tumours are consistent with a TKTL1-based, lactobacillae-like glucose metabolism in tumours. Which of the altered enzymatic activities leading to these metabolic changes can be exploited for future cancer therapies? The most effective way to inhibit tumour proliferation should be to block the generation of energy for tumour growth. Metabolic control analysis and inhibition of transketolase enzyme reactions have already shown that tumour proliferation can be inhibited by anti-transketolase approaches (


Pharmacokinetic studies after intraperitoneal administration showed that the serum level of AlPcS2a fitted a biexponential model . The tumour concentration was roughly constant up to 48 h, although fluorescence microscopy showed that the drug location was initially mainly vascular, but became intracellular by 48 h. To compare PDT with PCI, 48 h after intraperitoneal injection of 10 mg kg−1 AlPcS2a, and 6 h after direct intratumour injection of 50 μg gelonin (PCI) or a similar volume of phosphate-buffered saline (PDT controls), tumour-bearing animals were exposed to red light (150 J cm−2). Complete response was observed for more than 100 days in 50% of the PCI tumours but only 10% of the PDT tumours (P<0.01). In tumours examined histologically 4 days after light delivery, the depth of necrosis was 3–4 mm after PDT, but 7 mm after PCI. The deeper effect after PCI demonstrates that the light fluence needed to kill tumour is less than with PDT. We conclude that PCI with gelonin can markedly enhance the effect of PDT on this type of tumour and may have a role clinically as an adjunct to surgery to control localised disease.Photochemical internalisation (PCI) is a technique for releasing biologically active macromolecules from endocytic vesicles by light activation of a photosensitiser localised in the same vesicles of targeted cells. This study investigated the PCI of the toxin gelonin as a way of enhancing the effect of photodynamic therapy (PDT) on a human malignant fibrous histiocytoma transplanted into nude mice using the photosensitiser disulphonated aluminium phthalocyanine (AlPcS These p2a) was provided by Frontier Scientific . A stock solution of 2 mg ml−1 in phosphate-buffered saline (PBS) was kept at −20°C until use. Gelonin was purified from Gelonium multiflorum by Dr Gowsala Sivam, Bastyr University, Seattle, USA.Disulphonated aluminium phthalocyanine with the sulphonate groups on adjacent phthalate rings (AlPcS3) in the left hind limb of nude mice . These mice were maintained under specific pathogen-free conditions, with food and water supplied ad libitum. Housing and all procedures involving animals were performed according to protocols approved by the institutional animal care and use committee, in compliance with the Norwegian Animal Research Authority's guidelines on animal welfare and the guidelines of the UK Co-ordination Committee on Cancer Research (UKCCCR). To minimise the number of animals required, not all combinations of values were studied in the treatment group. Three animals per group were used for the serum and tissue analysis. The tumour size was measured by caliper in two dimensions twice a week and the volume calculated by the formula 0.5 × (length × width2). Treatment commenced at a mean volume of 100 mm3 (s.d.±25 mm3). Animals were killed by cervical dislocation if the volume reached 1000 mm3.A human malignant fibrous histiocytoma (MFH) xenograft (TAX1), established in 1988, was provided by Professor Ola Myklebost, Department of Tumour Biology at the Norwegian Radium Hospital, Oslo. It was derived from tissue obtained during resection after local relapse. The tissue was propagated by implantation of fragments . For calibration, measurements were made on tissues extracted from animals not given AlPcS2a, but to which known concentrations of AlPcS2a had been added.Animals injected with 10 mg kg2a were mounted in Tissue Tek II embedding compound , and 8 μm sections were cut with a cryostat microtome and mounted on clean glass slides. Fluorescence microscopy was undertaken using a Nikon Eclipse E800 microscope with a 100 W mercury lamp and fluorescence images were made with a highly light-sensitive thermo-electrically cooled charge-coupled device (CCD) camera , with a resolution of 1344 × 1024 pixels and a dynamic range of 16 bits per pixel. The equipment used a 330–380 nm excitation filter, a beam splitter and a 590 nm long-pass emission filter. The same lens (× 20) and integrated exposure time (2 s), known to cause less than 5% photobleaching of the dye fluorescence, were used throughout the study. The neighbouring tissue sections were stained with haematoxylin and eosin (H&E) and transmission images made with a colour CCD camera  to determine the exact microscopic distribution of the dye in the tumour tissues.The frozen tissue blocks for localisation of AlPcS2a (10 mg kg−1) and immediately placed in the light tight chamber of an XFO-6 fluorescence option system equipped with a CCD camera . The peak AlPcS2a excitation wavelength was 675 nm and emission was measured at 694 nm with a Cy 5.5 filter set, 1 s exposure time and a field of vision (FOV) of 25 cm2. Fluorescence emitted from the tumour tissue was used to generate a false colour image using LivingImage Software (Xenogen Corp.).For whole body images, four animals were killed 48 h after i.p. injection of AlPcSμg (25 mg kg−1 b.w.) gelonin dissolved in PBS (pH 7.2) and were killed 10 min, 1, 6, 24 or 48 h later. Control animals received no gelonin. At post mortem, the tumours were excised, snap frozen in liquid nitrogen and freeze-fractured with a Bio-Pulverizer  cooled thoroughly with liquid nitrogen. Then, tissue lysate was prepared by homogenisation in a modified RIPA buffer ) for 1 h at 4°C. Subsequently, tissue and cell debris were removed by centrifugation and the protein concentration of the supernatant was determined with a modified Lowry assay . The tissue lysates were boiled for 5 min in 1 × SDS sample buffer , separated by SDS–PAGE (100 μg protein well−1) followed by overnight transfer to a PVDF membrane . The membrane was then blocked in a TTBS buffer containing 5% dry-milk and afterwards probed for 1 h at room temperature (RT) with a rabbit polyclonal antibody  against gelonin. The membrane was subsequently washed 3 × 10 min (RT) with TTBS buffer followed by probing with a HRP-linked goat-anti-rabbit antibody in TTBS buffer containing 5% dry-milk for 1 h at RT. To visualise the specific bands of gelonin, the ECL Plus™ (Amersham) Western blotting detection kit was used. Chemifluorescence was detected by the Storm® gel and blot imaging system (Amersham).The abundance and stability of gelonin in the tumour tissue after intratumour (i.t.) injection was investigated by chemical extraction and Western blotting. Animals received a single i.t. injection of 50 2a+gelonin (no light) and 5: gelonin+light (no AlPcS2a). Groups 1, 2 and 4 were injected with 10 mg kg−1 i.p. AlPcS2a and kept in the dark for 48 h prior to light delivery (no light in group 4). Injection of 25 μl of gelonin  or PBS (groups 2 and 3) directly into the centre of the tumour was performed 6 h prior to delivery of red light from a 150 W halogen lamp  filtered through a 580 nm long-pass and a 700 nm short-pass filter . For light delivery, the mice were fixed in a holder and the area surrounding the tumour was completely covered with aluminium foil. The mice were then kept in subdued light for a further 6 days. Tumour size was documented twice a week. If there was apparent eradication, monitoring was reduced to once a week to look for any tumour recurrence. For histological assessment, separate animals were killed 4 days after light delivery and the tumours divided at the centre of the zone of necrosis into two parts in the plane parallel to the axis of light delivery. The specimens were fixed in 4% formalin for subsequent sectioning and staining with H&E. All histological sections were examined microscopically to document the extent of viable tumour tissue. The first cuts, representing the largest cross-section in the zone of necrosis were imaged by a scanner and hard copies printed for further estimation of the areas of viable and nonviable tumour.Tumour-bearing mice were allocated randomly to one of five groups: 1: PCI, 2: PDT, 3: no treatment, 4: AlPcS2a serum concentration kinetics was analysed by nonlinear regression of log concentration vs the log multiexponential decay with time. Significant differences between time points were assessed by one-way analysis of variance. Significant differences between PDT and PCI treatment and the histological findings after the treatment were assessed by t-test analysis.The AlPcS2a level in the mouse serum and tumour after i.p. administration. At 2 h after administration, the serum level was relatively high (∼600 ng ml−1), but this declined rapidly. The serum clearance did not follow a simple monoexponential decay, but fitted well to a biexponential two-compartment model described by the equation C=1420et−0.39+41.8et−0.026 (corr. coeff. R=0.9916). The model gives two half-lives of 1.8±0.58 and 26.7±2.9 h, correlating to elimination rate constants of 0.39±0.13 and 0.026±0.003 h−1. A three-compartment model did not improve the correlation with the observations.2a in the tumours was not significantly different between 2 and 48 h after administration (P=0.62) and was about 400ng g−1. Beyond 48 h, AlPcS2a was cleared from the tumour with an apparent elimination rate constant of 0.0124 h−1 (C=914et−0.0124). This constant is based on the AlPcS2a concentration in the tumour (ng g−1 tissue) and is thus biased by the simultaneous growth of the tumour. The tumour doubling time in these experiments was 147±6 h following the regression line V=85.5et0.047 mm3 (corr. coeff. R=0.997). The corrected elimination decay may thus be described as A=[914et−0.0124] × [0.0855et0.0047]=78et−0.0077, with the approximation that 1 g tissue equals 1 ml. The corrected elimination rate constant is 0.0077±0.0010 h−1, which indicates that the tumour growth compensates for only 0.0047/0.0124=38% of the tumour concentration decay.One-way analysis indicated that the amount of AlPcS2a was essentially eliminated from the serum within 24 h, while the concentration in tumour tissue was stable from 2 to 48 h after administration. Thus, the in vivo localisation of AlPcS2a was evaluated 2 and 48 h after administration by fluorescence microscopy. The cryosections obtained 2 h after sensitiser injection .A major requirement for PDT and PCI is that the photosensitiser should localise into the area to be treated. In addition, PCI requires the photosensitiser to be located in the same endocytic vesicles of parenchymal cells as the macromolecule of interest. Localisation in the blood or the tissue stroma will reduce the synergism. Our pharmacokinetic analysis indicated that AlPcSnjection  showed ater 48 h  showed anjection . Dissectnjection . The disA prerequisite for photochemical activation of gelonin is its stable presence in the tumour cells after intratumor injection. Gelonin may be excreted by the highly vascularised sarcomas or degraded by hydrolases. 3  was the same (31±6 days) in all control groups  and up to 11 days compared with PDT-treated animals (P<0.01)). There were no significant differences in tumour volume at the time of light delivery between the complete remission group and those that regrew later. All tumour-free animals were followed for 110 days.The time for tumours to reach 1000 mmP<0.001)  . MeasureP<0.001) . IntratuP<0.001)  or the tPhotodynamic therapy is a versatile treatment modality for cancer and is approved for use in the treatment of a variety of malignant and premalignant conditions  as long as they are not released into the cytosol by light activation (unpublished data). The tumour clearance rate for AlPcS2a, corrected for tumour growth indicates a half-life of 90 h (56 h uncorrected). This stable plateau of photosensitiser concentration in tumour tissue for 1–2 days after administration followed by clearance over several days has been reported for several photosensitisers  compared with PDT alone (light and AlPcS2a) or gelonin alone. The 50% complete response rate observed after PCI correlated with necrosis reaching a depth of 7 mm and covering a mean of more than 97% of the cut surface of tumours examined 4 days after light delivery. After PDT, the depth of necrosis was no more than 3–4 mm. This may indicate that tumour eradication is related to a threshold number of tumour cells surviving the treatment, and that this threshold may be around 2.5%. In this study, the gelonin was given by direct i.t. injection, so it is difficult to know exactly where it was located, but it may also be possible to administer it systemically, when one could be more confident that it reached all the cells in the target area. Our findings also provide evidence that PCI using gelonin effectively reduces the light fluence needed to obtain a cytotoxic effect inside tumours compared with PDT alone (Our results showed an increased survival time in tumour-bearing animals using the combination of the photoactivation of AlPcSThis study was undertaken to investigate the possibility of using PCI in the treatment of sarcoma patients before, during or after surgery, particularly to reduce the incidence of local recurrence. At present, 50% of patients die within 5 years due to local relapse or metastases . Chemoth


Investigations were carried out on two lines of human melanomas  xenografted in nude mice. The tumours were characterised by a similar vascular supply but showed a pronounced difference in the rate of volume growth and in the radiobiologically hypoxic fraction. The distribution of ATP, glucose and lactate in the tumours was investigated using quantitative bioluminescence and single photon imaging. Concentrations of the metabolites were obtained as global values for the entire tumour mass, in regions with densely packed, structurally intact tumour cells ('viable zones'), in areas with necrosis, stromal cells and fibrous material ('necrotic zones') and in adjacent normal tissue. In all melanomas investigated glucose concentrations were significantly lower and lactate concentrations were significantly higher than in normal tissue. In contrast, no significant differences for ATP were detected. ATP and glucose concentrations were significantly less in necrotic than in viable tumour zones, whereas lactate concentrations were nearly equal in these tumour parts. Corresponding results were obtained in central versus peripheral tumour zones. There was no dependency of global or regional metabolite concentrations on tumour size within the volume range 110-1470 mm3. Based on this lack of dependency, metabolic concentrations were averaged over the whole tumour size range. Metabolite concentrations were not significantly different either globally or regionally between the two tumour entities investigated, a finding which held true for all three metabolites registered. Thus, metabolite distributions apparently mirror the similarity in vascularity of MF and EE melanomas rather than reflecting intrinsic properties with regard to tumour growth rates or susceptibility to radiation.


Time courses for tumour growth, intracellular levels of FdUMP, thymidylate synthase (TS) activity, and 5-FU in RNA were also determined, and the effects of a 9.5-min period of carbogen breathing, starting 1 min before drug administration, on MRS-detected 5-FU metabolism and tumour growth curves were examined. Both tumour variants generated MRS-detectable 5-FU nucleotides and showed similar initial growth inhibition after treatment. However, the growth rate of C26-B tumours returned to normal, while the sensitive C26-10 tumours, which produced larger fluoronucleotide pools, still showed moderate growth inhibition. Carbogen breathing did not significantly influence 5-FU uptake or fluoronucleotide production but did significantly enhance growth inhibition in C26-10 tumours. While both tumour variants exhibited incorporation of 5-FU into RNA and inhibition of TS via FdUMP, clearance of 5-FU from RNA and recovery of TS activity were greater for the insensitive C26-B line, indicating that these processes, in addition to 5-FU uptake and metabolism, may be important determinants of drug sensitivity and treatment response.Following an i.p. dose of 150 mg kg Colorectal cancer is the third most common cancer in the Western world. Fluoropyrimidine chemotherapy with agents such as 5-fluorouracil (5-FU) is the main method of treatment, but is effective in only a small percentage of patients. More insight into the pharmacokinetics and intracellular metabolism of 5-FU in tumour tissue is essential for a better understanding of the variability of patient response and for the development of improved therapy protocols.19F magnetic resonance spectroscopy (MRS) is a powerful method for assessing these aspects in vivo since it allows the noninvasive detection and quantitation of 5-FU and its metabolites in tumour during therapy. Such experiments have been described for a number of animal models  were also examined. Furthermore, we have studied the effect of a short carbogen breathing period on the efficacy of 5-FU treatment for these two tumour variants since carbogen breathing causes vasodilatation and, therefore, may increase tumour perfusion and enhance delivery of 5-FU (The present study used −1). Carbogen gas (95% O2/5% CO2) was obtained from Hoekloos .5-FU was obtained from Teva  as a saline solution (50 mg ml3) was estimated from three orthogonal diameter measurements  as volume=0.5(xyz)  while C26-10 tumour volumes were 0.58 ± 0.29 cm3 (n=24). Mice were divided into groups based on estimated tumour volume, and stratified randomisation was carried out over the different treatment groups. All experimental procedures in the following sections were approved by the local ethics committee for the use of animals according to the standards required by the UKCCR guidelines , two groups of tumour-bearing mice  were prepared for 5-FU therapy and 19F MRS as follows. Each mouse was canulated i.p. under inhalation anaesthesia using a mixture of enflurane (1.5%), oxygen (29.5%), and nitrous oxide (69%) and mounted in the magnet bore of the MR spectrometer. Core temperature was monitored with a rectal temperature probe and maintained under anaesthesia at 37°C by means of a regulated warm water blanket. 5-fluorouracil was administered i.p. as a 150 mg kg−1 bolus by injection of 0.1 ml g−1 body weight of 5-FU (1.5 mg ml−1) in standard saline over a period of 20 s.At days 11–12 after implantation (tumour volumes in the range 0.07–1.77 cmn=11) and C26-10 (n=9) tumours. The animals were prepared for MRS as above, but 1 min prior to 5-FU administration the nitrous oxide and oxygen components of the anaesthesia were replaced with carbogen (2 l min−1). After administration of the 5-FU bolus, carbogen breathing was continued for an additional 8.5 min. The carbogen gas was then replaced by oxygen/nitrous oxide for the remainder of the experiment.A second, analogous series of experiments including carbogen breathing was performed with separate groups of mice bearing C26-B  After 1 min of carbogen breathing, the oxyhaemoglobin and deoxyhaemoglobin concentrations in human glioma xenografts were found to be close to their final steady-state values  over the time period day 0 to day 28 (as % of weight on day 0), the last day of the experiment, at which point the mice were killed by cervical dislocation.which provided a measure of the increase in doubling time caused by treatment  and a SMIS electronics console. A home-built, two-turn surface coil with an internal diameter of 13 mm, tuneable to 1 H or 19F, was placed around the s.c. tumour located in the right flank. The sensitive volume of the coil was centred on the tumour tissue. Magnetic field homogeneity was adjusted by shimming on the tissue H2O signal to reach a line width of less than 70 Hz (0.4 ppm).19F MRS, a simple pulse-acquire sequence was used with a spectral width of 10 kHz, a time domain of 4 K points, a rectangular radiofrequency pulse of length 20 μs (ca. 70° flip angle at the centre of the surface coil), and a repetition time TR=0.5 s. A flip angle β=70° is optimal in repetitive pulsing experiments for TR/T1=1.07, giving >96% of the maximum possible signal-to-noise ratio. The 19F T1 values for 5-FU and its metabolites have not been measured under our in vivo conditions, but are expected to lie in the range of ca. 0.12–1.7 s, measured at 1.5 T , and B1  is proportional to the local detection sensitivity. The total signal will be the sum over all detectable voxel signals and will depend on tumour size, metabolite concentration distribution, as well as the spatial dependence of the saturation and sensitivity factors. For a 70° flip angle, saturation factors  will be 0.72–1.0 for TR/T1=1 (expected to apply for fluoronucleotides) or could be as low as 0.35 for the extreme condition TR/T1=0.3 (corresponding to T1=1.7 s in our experiments).is the local saturation factor , and signal vs time for a given metabolite should be roughly proportional to an effective mean tissue concentration vs time if saturation and sensitivity factors and the distribution of the metabolite relative to the surface coil do not change dramatically.Thus, in any given 1 H decoupling was not used since the in vivo linewidths were much larger than 3JHF (5 Hz) for the fluorouracil moiety. The time-domain 19F MR data were stored as sequential 8.5-min blocks (1024 transients per free induction decay (FID)), beginning at the moment of 5-FU injection and continuing over a period of 2 h.1 H images and estimated that nearby liver tissue represented less than 5% of the detected volume. The absence of a significant contribution from liver or the intraperitoneal volume to the 19F MR signals was further confirmed by two 19F MRS measurements performed on animals without tumour but treated as described above. In these cases, very little signal from 5-FU or its metabolites was detected.To check the localisation of the MR-sensitive volume, we used the same experimental set-up to obtain 1 H MR spectra were recorded at 180.130 MHz before and after 19F MR acquisition with the rf coil still tuned to 19F . These conditions are similar to those used for 19F . The resulting signal from tissue water will represent a complex sum of product terms for each voxel, as described above for the 19F measurements. Since the flip angle and TR/T1 parameters are similar for the 19F and 1 H measurements, the tissue volumes ‘seen’ by the two experiments should be similar . Assuming tissue water concentration is roughly constant, then the 1 H signal intensity should increase with tissue volume (tumour size) in the same manner as the 19F signal will increase with tumour size if the detected metabolite has a relatively uniform distribution with constant mean concentration. Therefore, the 1 H signal integral was used to normalise the 19F signal integrals obtained from different tumours and to provide a crude form of compensation for differences in signal intensity caused by differences in tumour volume alone. The 1 H MR spectra were recorded before and 2 h after carbogen breathing to avoid effects of carbogen on the amplitude or integral of the water 1 H signal (relaxation effects due to change in paramagnetic oxygen concentration).19F time-domain data (17-min acquisitions) were added and multiplied by an exponential window function (line broadening of 20 Hz) before Fourier transformation to the frequency domain. The 19F MR resonance frequency observed for 5-FU in vivo was defined as the reference point for all chemical shift measurements . Quantitative analysis of the 19F and 1 H MR spectra was performed by fitting the time-domain signal using the software package MRUI  and α-fluoro-β-alanine (FBAL: −19.2 ppm). All 19F signal integrals were normalised by dividing by the mean value of the H2O signal integral obtained from the two 1 H reference spectra. The normalised 19F MR signal integrals were then considered to represent mean relative metabolite concentrations per unit volume of tissue (averaged over the sensitive volume detected by the surface coil) and, for a given metabolite, can be compared across all animals and groups for all tumour volumes. This technique assumes that the water 1 H signal and the metabolite 19F signals exhibit the same dependence for intensity vs tumour volume, which should be a reasonable assumption if both tissue water and 5-FU metabolites have similar distributions, as discussed above. No corrections were attempted for possible differences in 19F T1 and saturation factors for the various metabolites, which may influence their relative signal integrals for a given tumour spectrum. The tissue half-life (t1/2) of 5-FU was estimated from the 19F MRS time course as the time required for 5-FU to decrease from its maximum concentration to half that value.Relative tissue levels (in arbitrary units) for 5-FU (F), total detectable anabolites , and total catabolites (C) were estimated from the signal integrals for 5-FU, the spectral region 4.2–5.2 ppm corresponding to FUMP, FdUMP, FUTP, etc., and the region −16 to −20 ppm corresponding to the major catabolites −1 bolus, i.p.) as described above for the MRS and growth studies. Tumours were removed at various time points after treatment and immediately frozen in liquid nitrogen. Frozen tumours were pulverised , alkaline phosphatase (dephosphorylation), and uridine phosphorylase (breakdown of fluorouridine to 5-FU) resulted in the stepwise degradation of RNA to give 5-fluoro-uridine-3′-monophosphate (3′-FUMP), 5-FUrd, and finally 5-FU, which was analysed by GC–MS  in order to avoid the development of an unacceptably large tumour burden during the course of the experiment when treatment was ineffective. However, in the volume range tested, no significant differences in TS levels were found, as long as the tumours were in the exponential growth phase . The two-sample Wilcoxon rank-sum test was used to evaluate differences in t1/2 for 5-FU . The parameters 19F MR signals were observed. The signal F at 0 ppm on the chemical shift scale represents unmetabolised 5-FU; it reached a maximum intensity within the interval 0–17 min after drug administration and decreased thereafter. The cytotoxic anabolite pool derived from 5-FU  gave one composite peak (A) at ca. 4.8 ppm, which could be detected in the second or third measurement block (8.5–25.5 min) and increased to a maximum plateau level after ca. 60–80 min when 5-FU was no longer detectable. Higher levels of anabolites (A) were generally observed for the 5-FU-sensitive C26-10 tumours (In  tumours . CatabolCmax as well as the AUC were significantly higher for the 5-FU-sensitive C26-10 tumour compared to C26-B (P<0.001), with or without carbogen breathing. In contrast, Cmax and AUC for catabolites and 5-FU as well as the half-life t1/2 of 5-FU were not significantly different for the two tumour types (P>0.2). Treatment with carbogen had no effect on the mean values for the parameters of 5-FU metabolism for either tumour variant , but resulted in a significant reduction in the range of Cmax and AUC for catabolites.The quantitative results from the MR experiments were determined by analysis of the original data with 8.5-min time resolution and are summarised in P>0.05). On the other hand, the initial incorporation of 5-FU into RNA at day 2 or 3 after treatment was about a factor of 3–4 lower in C26-10 compared to C26-B (P<0.001 on day 3). However, at day 7 or 10 there was a more rapid decrease in FU-RNA in the insensitive C26-B tumours to values less than those observed for C26-10, and residual FU levels in C26-B were about one-sixth the values for C26-10.Separate groups of tumour-bearing mice were given the same 5-FU treatment as for the MRS studies (day 0=11–12 days after implantation), and tumours were excised at various times thereafter. Extracts of the excised tumour tissues were used to determine the time courses for metabolites and TS enzyme activity presented in P<0.001). For both tumours, TS activity decreased significantly 1 day after 5-FU administration, rebounded to about twice the control level by day 7, and finally decreased towards control levels on day 10. The relative inhibition of TS (compared to controls) at day 1 was significantly higher for C26-B (factor 8) vs C26-10 (factor 2.6), while the relative degree of overshoot at day 7 was similar.Thymidylate synthase catalytic activity in untreated C26-10 control tumours was found to be about half the level in C26-B controls . Following 5-FU treatment, the number of binding sites for both tumour types decreased by about a factor of 4 at day 1 and rebounded with the same overshoot behaviour at day 7 as observed for TS activity. The number of binding sites for C26-10 relative to C26-B remained at 60–80% over the 10-day time course.Histological examinations of six individual untreated tumours of C26-B and of C26-10 murine colon carcinoma were performed. Both variants were poorly differentiated adenocarcinoma with less than 15% necrosis on days 11–12 after implantation, and no clear differences in histology or vascularisation were observed.Normalised growth curves for s.c. C26-B and C26-10 tumours in Balb/C mice following i.p. bolus 5-FU treatment are shown in Figure 2P=0.048), but the growth delay factors were similar (2.52 vs 2.23).5-Fluorouracil treatment without carbogen led to moderate regression at day 4 for both tumour types, but growth continued again after day 7 with C26-B exhibiting the higher growth rate. Owing to 5-FU, the doubling times calculated from day 0 increased to 10.2 days , giving a GDF>5.8. Tumour regrowth began only after an average of 17 days. Following the single bolus 5-FU treatment, complete tumour remission was observed only for two out of 26 C26-10 tumours (one with and one without carbogen).When carbogen was applied during treatment, there was no significant change in the initial response of C26-B tumours , but there was a moderate decrease in the mean growth rate after day 11 for the carbogen group. A significant effect of carbogen was observed, however, for the 5-FU-sensitive C26-10 tumours (group 4) with the median doubling time increasing to >28 days (The mean percentage weight loss (WL) over the time period days 0–28 was 6% for the C26-B tumour-bearing animals treated with 5-FU alone and 12% in the other three groups. These values were considered to be within acceptable limits.19F MRS results (0–2 h time course) for two variants of the C26 tumour line with different sensitivities to 5-FU, along with biochemical data derived from tissue extracts (time courses from 2 h postdrug to up to 10 days). Our 19F MRS results show that the more sensitive C26-10 tumour exhibits a significantly higher conversion of 5-FU to fluoronucleotides (cytotoxic anabolites) compared to the insensitive, more rapidly growing C26-B tumour  or its half-life (t1/2). Therefore, these parameters cannot account for the higher anabolite levels observed in C26-10 compared to C26-B. Differences in catabolism of 5-FU in tumour via dihydropyrimidine dehydrogenase (DPD) can also be ruled out since DPD was not detectable in either tumour variant  compared to C26-10, and both 5-FU-RNA and free 5-FU levels were lower in C26-B at days 7 and 10. This ability of C26-B tumour cells to clear more rapidly 5-FU from RNA may contribute to the insensitivity of this cell line to 5-FU therapy and the return to a normal growth rate after day 7  and higher basal TS catalytic activity  prior to therapy (presumably improved by carbogen) and a high level of fluoronucleotides produced following a 5-FU bolus were associated with tumour regression. However, we do not have appropriate 31P-MRS data to address this point for C26. It is known that the effect of carbogen may depend on tumour characteristics such as type, size, vascularisation, perfusion, and pH (vs i.v. drug administration (We note that it has been demonstrated for a rat tumour model (in vivo 5-FU uptake and anabolic conversion as well as recovery from treatment via clearance of 5-FU from RNA and induction of TS. These differences are consistent with and probably responsible for the observed differences in sensitivity to 5-FU treatment. These findings concerning possible mechanisms or sources of drug resistance may be of immediate clinical relevance since cancer patients exhibit a wide range of sensitivity to fluoropyrimidine therapy.In conclusion, we provide quantitative evidence for differences between two variants of the C26 murine colon carcinoma in terms of


The sensitivity and negative predictive value of tumour budding alone for lymph node metastasis were superior to those of lymphatic invasion alone. Furthermore, the specificity and positive predictive value of the combination of either lymphatic invasion or tumour budding were superior to those of lymphatic invasion alone. Tumour budding detected immunohistochemically by using CAM5.2 is a newly found risk factor for lymph node metastasis and may help to avoid oversurgery in the future.In the past few years, tumour budding at the invasive margin has been reported as a new risk factor for lymph node metastasis in advanced colorectal cancers, but it is sometimes difficult to detect tumour budding in submucosal colorectal cancer by haematoxylin and eosin staining. We immunohistochemically examined tumour budding at the deepest invasive margin of 56 surgically resected submucosal colorectal carcinomas using anticytokeratin antibody CAM5.2, furthermore checked by AE1/AE3, and determined the relation between tumour budding and clinicopathological factors. Moreover, we used the monoclonal antibody D2-40 for immunohistochemistry to detect lymphatic involvement. Tumour budding was detected in 42 cases (75.0%), and the budding-positive group showed a significantly higher rate of lymph node metastasis (including isolated tumour cells) (16/42  Recently, endoscopic polypectomy or endoscopical mucosal resection has been performed extensively for the treatment of colorectal adenomas, and intramucosal and submucosal carcinomas  in various gastrointestinal and biliary cancers  of submucosal cancer using CAM5.2. The monoclonal antibody D2-40, which has been developed against a testicular oncofetal antigen, has been demonstrated to recognise lymphatic vessels in the breast, colonic carcinoma and head and neck squamous cell carcinoma  (diluted 1 : 3) and AE1/AE3  (diluted 1 : 150) immunostaining were cut from the deepest invasive specimen in order to examine the budding at the deepest invasive margin  (diluted 1 : 100) to visualise the endothelial cells of the lymphatic channels , from each of the paraffin blocks of the lymph nodes, which were used for CAM5.2 immunohistochemical examination as described below . For micrometastasis and ITCs analysis, we examined three serial sections, each 10 ed below .M sodium citrate buffer (pH 6.0), and heated in a microwave oven for three 7-min cycles (500 W). After washing twice in phospahte-buffered saline (PBS), endogenous peroxidase activity was inhibited by incubation with 0.3% hydrogen peroxide in methanol for 20 min. After three washes in PBS, nonspecific reaction was blocked by incubation at room temperature. Reagents for the subsequent step, biotinylated rabbit anti-mouse immunogloblin and SAB complex, supplied commercially (Histfine SAB-PO(M) kit, Nichirei, Tokyo, Japan) were used. The sections were incubated with anti-D2-40 antibody overnight at 4°C. Colour was then developed with diaminobenzidine solution. The sections were then lightly counterstained with a cocktail of Mayer’s/Lillie–Mayer’s haematoxylin and mounted. For CAM5.2 and AE1/AE3 staining, the sections were deparaffinised with xylene and dehydrated with 98% ethanol, and treated with 0.1% trypsin  in 0.1% calcium chloride (pH 7.8) at 37°C for 20 min before immunostaining; they were treated in the same way as with D2-40 staining, and counterstained with methyl green and mounted.The streptavidin–biotin (SAB) immunoperoxidase method was used. For D2-40 staining, the sections were deparaffinised with xylene and dehydrated with 98% ethanol, placed in 0.01 Tumour budding refers to microscopic microtubular cancer nests and undifferentiated cancer cells at the invasive margin of the carcinomatous lesion, as proposed by Tumour deposits within lymph nodes were classified according to the revised guidelines set by American Committee on Cancer (AJCC) . The characteristics of the 56 tumours are shown in vs 0/14; P=0.004) than the budding-negative group. Moreover, the former group showed a higher rate of lymphatic involvement  than the latter group, but not significantly. All 14 cases without tumour budding showed no lymphatic involvement or lymph node metastasis. There was no relation between tumour budding and venous involvement.Tumour budding was detected in 42 cases (75.0%) by CAM5.2 immunohistochemistry, and all cases detected by CAM5.2 were also positive for AE1/AE3 immunohistochemistry. No case with tumour budding was newly detected by AE1/AE3 immunohistochemistry. The sensitivity, specificity and predictive value of lymphatic invasion, tumour budding and the combination of these two factors for lymph node metastasis are shown in We were able to show that tumour budding is a newly discovered risk factor for lymph node metastasis in submucosal colorectal carcinoma. There is, therefore, a possibility that oversurgery for polypectomised submucosal colorectal carcinoma can be avoided by examining tumour budding.Tumour budding is defined as anaplastic thin cell cords or individual free cells, microtubular cancer nests or undifferentiated cancer cells , or upregulation of CD44 variant 6 through nuclear β-catenin activation contributed to the formation of budding cell at the invasive front. In the past few years, important molecular events concerning with tumour budding, such as the gain and loss of adhesion molecules, secretion of proteolytic enzymes, reduction of tumour proliferation and mutation of tumour suppressor genes, have been examined. In conclusion, our results indicate that tumour budding correlates with lymph node metastasis in submucosal colorectal cancers, and that this parameter is a useful indicator of the risk of lymph node metastasis in such cancers. Detection of tumour budding by CAM5.2 immunohistochemistry may help to avoid oversurgery in the future. A new study with a larger number of cases, especially in a prospective and multicenter setting, is necessary.


The relationship between two methods of assessing tumour oxygenation in vivo, namely oxygen electrode measurement and magnetic resonance spectroscopy (MRS) of the fluorinated 2-nitroimidazole SR-4554, was investigated. Using three tumour models (two sites), no linear correlation was observed between 19F retention index and pO2 parameters (r < or = 0.3). Substantial retention of SR-4554 (19F retention index > 0.5) was, however, associated with low tumour pO2 (% pO2 < or = 5 mmHg = 60%). Depending on the pO2 parameters used, SR-4554 administration was shown to produce either a significant or a non-significant increase in tumour oxygenation. We conclude that measurement of SR-4554-related compound(s) by 19F-MRS has the potential to detect clinically relevant levels of tumour hypoxia.