Tumour size and nodal involvement are the two main prognostic factors in breast cancer (BC). Their impact on the natural history of BC is not fully captured by analyses that ignore their quantitative nature.Data pertaining to 18 159 patients treated with primary surgery: 3661 at the Institut Gustave-Roussy  between 1954 and 1983, and 14 498 in the breast cancer registry in the Stockholm–Gotland Health Care region  between 1976 and 1999, were collected. The risks of distant metastases (DMs) and of nodal involvement were analysed according to tumour size with parametric models.P<10−10) in IGR and 0.61  in SG 1991–1999. Differences in tumour size explained the increased risk in IGR , but not the decreased risk in SG 1991–1999 . The relationship between tumour size and DM risk changed significantly during the 1990s.Using SG 1976–1990 as the reference group, relative risks (RRs) for DM were equal to 1.42 (95% CI: 1.29–1.56; Early diagnosis is sufficient to explain differences in the prognosis before 1990. After 1990, the use of adjuvant systemic therapies is the main reason for the reduction in DM. In developed countries, the incidence of breast cancer increased regularly from 1975 to 2000 . The conhttp://www.adjuvantonline.com)  system and of new prognostic tools such as Adjuvant! (ine.com) , are actAs the study period spans about 50 years, we also investigated the effect of the year of treatment and, consequently, of the effect of changes in management strategies  on the relationship between tumour size and the risks of nodal involvement and metastasis.This database includes 7688 consecutive patients with breast carcinoma treated at the Institut Gustave-Roussy  between 1 January 1954 and 31 December 1983. The database was updated every 2 years and last updated in 2003–2005.The Stockholm–Gotland Health Care region (SG) registry (Sweden) covers a population of about 1 million women (946 784 in 1999), and around 1000 new breast cancer cases are now registered annually. Follow-up is continuously updated. During the period spanning from 12 April 1976 to 31 December 1999, 21 700 breast cancer patients were included. The last follow-up date was 1 April 2005. The Stockholm mammography screening programme was started in 1989, inviting women between 50 and 69 years of age to have a mammography at 2-year intervals  and qualitative data . The logistic and complementary log–log regressions (or Cox's model when time to event was used as a continuous variable) led, respectively, to odds ratio and relative risk estimates, which are useful for assessing risks but not for understanding the mechanisms or the natural history of the disease. Probit regressions were used to estimate tolerance distributions, for example, distributions of tumour volume required to obtain a given proportion of patients with involved nodes, or a given proportion of patients with detectable metastases within 5 years of treatment.The tumour volume was calculated from the largest tumour diameter assuming spherical lesions. In the regression models, the quantitative variable used for tumour size was the logarithm of tumour volume. We analysed two end points: the number of involved axillary lymph nodes  and the occurrence of distant metastases during the 5 years after treatment.Axillary nodal involvement was analysed through regressions between tumour size and the number of positive axillary lymph nodes. The number of sampled nodes was introduced as a covariate with five categories .The relationship between tumour size and metastasis occurrence was analysed after grouping tumours by diameter in 1-cm increments. We estimated the 5-year cumulated proportion of patients with metastases, and the geometric mean tumour size in each group.We used the Kaplan–Meier method to estimate the proportions of patients with distant metastases. Only distant metastases registered as first events were counted as events. Patients who experienced a competing event first were censored at the date of the first event. Deaths were never counted as events: distant metastases followed by deaths were counted as an event at the time of the first distant metastasis; patients dying without distant metastases were censored at death. Local recurrences that occurred first were not counted as events; patients were censored at the time of the local recurrence, and we implicitly assumed that their risk of distant metastases was similar to that of women who had not experienced a local recurrence.Patients were distributed into three groups as follows : (1) allThe tumour volume corresponding to a 50% 5-year risk of metastases was calculated in each cohort. The number of involved nodes was analysed as a function of tumour size, the number of sampled nodes and the cohort. The tumour volume corresponding to 50% of tumours with involved nodes was calculated in each cohort (assuming a number of sampled nodes of between 7 and 9) and it was used as an indicator of the tumour's propensity to invade axillary lymph nodes.The total population (18 159 patients) was divided into 18 consecutive cohorts of patients (17 first cohorts including 1000 patients and the last cohort including 1159 patients) with no distinction between IGR and SG patients. The relationship between tumour size and the proportion of patients with metastases at 5 years was analysed with probit regression, assuming similar slopes in all cohorts.χ2 tests and the distributions of quantitative variables with non-parametric Kruskall–Wallis tests. The proportions of patients free of distant metastases according to the time since treatment were compared with log-rank tests. The statistical significance of the regression parameters was assessed with Wald tests. All the analyses were carried out using Statistical Analysis System  software.The distributions of qualitative variables were compared with This study was carried out by a collaboration between the Institut Gustave-Roussy , the Karolinska Institutet  and the Stockholm–Gotland Breast Cancer Group. The two institutes funded the sabbatical periods spent by SK in Stockholm.The three patient groups differed significantly for almost all study parameters . PatientP<10−10). The hazard functions for distant metastases were unimodal and the hazards reached a maximum during the second 1-year interval after treatment (P<10−10) in IGR 1954–1983 and 0.61  in SG 1991–1999.The incidence of distant metastases according to time since treatment  differedreatment . The hazvs 14.8% for an 18-mm tumour) between the two groups. Similarly, the odds ratio (logistic model) and relative risks (Cox's model) for distant metastases were not different from 1 when adjusted for tumour size used as a continuous covariate. It should be noted that when tumour size was categorised with a simple cutoff at 3 cm, the odds ratio and relative risk became significantly different from 1 . The relationships between tumour volume and the 5-year risk of distant metastases were not different between IGR 1954–1983 and SG 1976–1990 . SmallerP<10−10). The logistic regression and Cox models adjusted for tumour size were also consistent with a highly significant difference between the two groups (P<10−10). The tumour volume required for a 50% rate of metastases at 5 years was multiplied by >3 in SG 1991–1999 (>900 ml) compared with the other two groups (<300 ml).In contrast, the decrease in the 5-year risk of distant metastases between SG 1976–1990 and SG 1991–1999 was not explained by differences in tumour size. In the three regression models, the relationship between tumour size and the probability of distant dissemination changed significantly between the two periods in the Stockholm material (P<10−10), but was not different for SG 1991–1999 (P=0.60) (P=0.03) and smaller than the volume estimated for the second Stockholm period (P=0.01). However, even if the differences concerning axillary node involvement as a function of tumour size were statistically significant, their clinical importance is limited: the difference in the risks of nodal involvement adjusted for tumour size (estimated for an 18-mm tumour diameter) was only 2% (P=0.01) between SG 1976–1990 and SG 1991–1999. Similar results were obtained when logistic regression was applied for axillary node involvement.With SG 1976–1990 as the reference group, the risk of nodal involvement was significantly higher for IGR 1954–1983 ((P=0.60) . The tumThe 5-year probability of metastases decreased regularly according to the year of surgery . OverallP<10−10, P<10−10 and P<10−5, respectively), whereas the relationships between tumour size and axillary lymph node positivity and 5-year proportion of patients with metastases were not significantly different. All the conclusions regarding comparisons between IGR 1954–1983 and SG 1976–1989 are also valid for those between IGR and SG that were restricted to patients treated during the 1976–1983 period.We also performed a comparison between IGR and SG that was limited to the 5057 patients treated during the 1976–1983 period, which is common to the two centres: 1756 patients in IGR and 3301 in SG. The differences/similarities between these two populations were the same as that observed in the broader comparisons between IGR 1954–1983 and SG 1976–1989. The tumour size, the proportion of patients with involved nodes and the 5-year proportion of patients with metastases were significantly larger in IGR 1954–1983 (P<10−10). The issue was to determine to what extent this variation was attributable to differences in tumour malignity, or to earlier diagnosis or to better treatment strategies. Our main findings were (a) the constancy of the relationship between tumour size and nodal involvement, (b) no significant differences in the relationship between tumour size and the 5-year risk of distant metastases for IGR 1954–1983 and SG 1976–1990 and (c) a change in this relationship after 1990 in the Stockholm database.This study included patients treated in two European countries, over a 50-year period. The most striking result is the regular decrease in the risk of developing distant metastases according to the year of surgery. For a patient treated in 1999, the risk of metastases was about one-third than that of a patient treated in the 1960s . However, the relationship between tumour volume and risk of metastases was not significantly different between the two groups (P=0.07), nor was the relationship between tumour volume and nodal involvement (P=0.03). When adjusted for tumour size, the relative risk of metastases was not significantly different between the two groups . This finding implies that differences in the initial tumour volume, related to early diagnosis in SG, explain most of the difference in prognosis between the two groups of patients.The risk of metastases was significantly higher in IGR 1954–1983 than in SG 1976–1990 , whereas the relative risk adjusted for the tumour size category (cutoff at 3 cm) was equal to 1.29 . This example emphasises the importance of analyses using quantitative data and suggests that some recent results, such as a significant independent effect of mammography, observed by We found only one study evaluating the impact of early diagnosis using quantitative estimates of tumour size , the 5-yThe difference between the two consecutive groups of patients from the Stockholm–Gotland region (SG 1976–1990 and SG 1991–1999) was not explained by differences in the initial tumour volume, or by a change in the intrinsic malignity of tumours. This difference can only be attributed to a decrease in the viability of metastases due to adjuvant therapy. The more frequent use of endocrine therapy – mostly tamoxifen– which increased from <20% to about 80% in the few years after 1990 (In conclusion, our results support the conclusions of the CISNET group and extend their findings. In our clinical series, early diagnosis is sufficient to explain the differences in the prognosis before 1990. After 1990, the generalisation of adjuvant systemic therapy, mostly tamoxifen, is the main reason for improved disease control and better use of adjuvant chemotherapy is likely to improve outcomes further.


This study investigates the effects of the anti-metabolite 5-fluorouracil (5-FU) on the human colon cancer line HT29 (10(7) cells per dose) grown subcutaneously in severe combined immunodeficient (SCID) mice. The efficacy of 5-FU was quantitatively evaluated by comparing the tumour weight, mitotic and apoptotic tumour cell indices and the expression of the Ki-67 nuclear antigen in drug-treated animals and control animals. The tumour cell carbohydrates were assessed using a lectin panel. A significant reduction in the tumour weight was found 4 days after initial 5-FU treatment. 5-FU treatment reduced the percentages of mitoses but increased the apoptotic index in the tumour cells. In addition, 5-FU induced an increase in the signet ring cell population and an increased binding for lectins specific for N-acetylgalactosamine and galactose. However, the vast majority of signet ring cells were negative for Ki-67. The results of this study indicate that continuous treatment with 5-FU for 4 days targets metabolic processes relevant for both cell division and apoptosis. The relative increase in the signet ring population can be explained by the fact that the more proliferation-active stem cell population of the tumour is the primary target of the therapy. The lectin-binding patterns reflect these changes and are therefore differentiation linked.


The goal of this study was to investigate the efficacy of photodynamic therapy (PDT) of a single tumour growing intraperitoneally. For this purpose the CC531 colon carcinoma, implanted in an intraperitoneal fat pad of Wag/RijA rats, was treated with intraperitoneal photodynamic therapy (IPPDT) using Photofrin as the photosensitiser. Two illumination techniques have been compared. An invasive illumination technique using Perspex blocks to illuminate 30 cm2 of the lower abdomen gave a significant delay in tumour growth with 25 J cm-2 applied 1 day after Photofrin. A minimally invasive illumination technique using a balloon catheter to illuminate 14 cm2 resulted in an equivalent growth delay with 75 J cm-2. The route of administration of the photosensitiser did not influence regrowth times of the tumour. Mitomycin C (MMC), a bioreductive agent, was used to exploit the known PDT-induced hypoxia. The combination of IPPDT with MMC resulted in an increased tumoricidal effect. In conclusion, IPPDT led to a significant growth delay for a single tumour implanted intraperitoneally and repetition of the PDT treatment was possible using a minimally invasive illumination technique. Repeated treatments resulted in increased tumour response.


Specific monoclonal antibody (mAb) 201B was used to deliver greater than 30% of the injected dose to lung where tumours had developed due to intravenous injection of cells. Specific213Bi-mAb 201B treatment of BALB/c mammary carcinoma EMT-6 tumours in lung resulted in a dose-dependent destruction of tumours and an extended lifespan of treated animals relative to controls. Significant reduction of lung tumour burden was noted in animals treated with 0.93 MBq injected dose or as little as 14 Gy absorbed dose to the lung. Animals treated with higher doses (2.6–6.7 MBq) had nearly complete cure of lung tumours but eventually died of lung fibrosis induced by the treatment. Four other tumour cell types were studied: murine Line 1 lung carcinomas in syngeneic BALB/c mice, rat IC-12 tracheal carcinoma growing in severe combined immune deficient (SCID) mice, and two human tumours – epidermoid carcinoma A431 and lung carcinoma A549 – growing in SCID mice. In all cases, the number of lung tumour colonies was reduced in animals treated with specific, labelled mAb relative to those in animals treated with control213Bi MAb or EDTA complexed213Bi. Tumours treated in immunodeficient SCID mice were partially destroyed or at least retarded in growth, but ultimately regrew and proved fatal, indicating that an intact immune function is necessary for complete cure. The data show that the short-lived α-particle emitter213Bi can be effectively targeted to lung blood vessels and that tumour cells growing in the lung are killed. The mechanism may involve direct killing of tumour cells from α-particle irradiation, killing through destruction of blood supply to the tumour, or a combination of the two. © 1999 Cancer Research CampaignA model system has been used to test the efficacy of vascular targeting of α-particle emitter


Serum cytotoxicity was first detected after 2 weeks of tumour growth, reaching a peak at 4 weeks and then declining. Inhibitory activity was found only in the sera of animals with advanced tumours. Anti-tumour antibody either in the sera or bound to tumour cells was not detected by immunofluorescence techniques. No evidence of general immunological debilitation was found, the animals showing normal immune responses to sheep erythrocytes and killed Brucella abortus organisms throughout tumour growth. Serum inhibitory factors may be responsible for the decline in anti-tumour immunoreactivity and the local lymphocyte anergy observed in tumour bearing hosts.The development of serum factors by Wistar rats during the growth of a syngeneic squamous cell carcinoma has been investigated to clarify the nature of the local lymphocyte anergy reported previously in this system. Sera from tumour bearing animals were tested for cytotoxicity against tumour cells by


Novel antiangiogenic agents currently being developed may ultimately be more effective against solid tumours and less toxic than cytotoxic chemotherapy. As a result of the early clinical trials of angiogenesis inhibitors, investigators are beginning to appreciate the complexity of targeting angiogenesis and the realisation that developing clinically useful antiangiogenic therapy will be more challenging than originally thought. It is now apparent that new methods and surrogate markers to assess these agents' biological activity are crucial for their successful development. This review summarises the currently available clinical data on the development of surrogate markers of angiogenesis inhibitors. Many anPreclinical studies suggested that angiogenesis inhibitors are cytostatic (growth-delaying) rather than cytotoxic. Traditionally, the maximum tolerated dose of a cytotoxic agent (determined by its dose-limiting toxicity) provided an estimation of the active dose range for subsequent clinical studies  that are recruited from the bone marrow during tumour neovascularisation  after initiation of treatment might provide an early indication of antiangiogenic activity before clinically demonstrable reductions in tumour size. Elevated levels of angiogenic growth factors, proteases, and endothelial adhesion molecules have been detected in sera of patients with malignant disease  and the basement membrane during angiogenesis include the family of matrix metalloproteinases (MMPs) . Proteolvs those with stable disease  of action of the angiogenesis inhibitor or the disease under study. In a Phase I study of tetrathiomolybdate  can provide early proof of whether the biologic agent has successfully reached its hypothesised target. Some of the most compelling studies involved quantitative analysis by laser scanning cytometry (LSC) . In contrast, preclinical studies suggested that these small molecules had great potential, as they induced significant levels of apoptosis in endothelial cells, consequently inhibiting tumour growth  . In addition, LSC-mediated analysis of additional surrogate markers hypothesised to be effected by endostatin including nuclear hypoxia-inducible factor-1α and endothelial cell Bcl-2 levels peaked at similar doses of endostatin but failed to reach statistical significance. Importantly, two patients treated at intermediate doses of endostatin had minor antitumour responses  and tumour metabolism [18F](FDG-fluorodeoxyglucose) 28 and 56 days after endostatin therapy . Importantly, the 95% confidence interval identified for blood flow overlapped the OBD (250 mgm2d) determined from the tumour biopsy studies. Thus, integration of surrogate marker data along with imaging studies was crucial for assessing the OBD of recombinant human endostatin.Routine radiographic imaging techniques  are useful for conventional measurement of disease and evaluating the effect of a biologic agent on tumour size  after initiation of therapy may provide an assessment of optimal antivascular activity. Indeed, LSC-mediated analysis of apoptosis in tumour cells 48 h after initiation of treatment has proven useful for predicting clinical response to cytotoxic therapies in breast cancer (


We investigated the metastasizing capacity of spontaneous lung metastases from the MN/MCA1 and mFS6 sarcoma, the B16 melanoma and colon 26 carcinoma. Spontaneous metastases at other visceral organs  from the M5076/73A (M5) ovarian carcinoma and colon 26 carcinoma were also studied. Tumour cells from individual spontaneous metastases were used immediately after isolation from the normal parenchyma  and/or after 1 s.c. passage in syngeneic mice . Spontaneous metastases were examined for all tumours and their secondaries after i.m. or s.c. inoculation of tumour cells; artificial lung colonies were measured after i.v. injection only of cells from the primary mFS6 and MN/MCA1 and B16 or their spontaneous metastases. Individual spontaneous metastases were to some extent heterogeneous in their metastatic potential, a minority of the secondaries having greater or lesser metastatic capacity than the appropriate primary. Overall, tumour cells from spontaneous metastases did not show greater metastasizing capacity than primary neoplasms, nor was there evidence that metastases from specific organs (e.g. spleen and kidney) tended to home to the specific anatomical sites from which they were originally isolated. These observations in a series of murine tumours of different histology, transplantation history and pattern of metastasis, do not support the hypothesis that metastases are the ultimate expression of strong selection of variant cells with greater intrinsic metastatic potential, pre-existing within the primary tumour.


The following funding information was omitted: FW acknowledges financial support from the German Ministry for Education and Research (BMBF) via the Bernstein Center for Computational Neuroscience (BCCN) Göttingen, Germany, under Grant No. 01GQ0430.


Stimulation of CFE by CS occurred in adrenalectomized rats but was inhibited by treatment of rats with an anti-inflammatory steroid, dexamethasone. CFE was stimulated by CS in tumour immunized rats; the inflammatory state did not prevent the expression of immunity but “rescued” a proportion (approximately 20%) of the injected tumour cells from immunodestruction in the lungs. A higher proportion of tumours grew in the paws of rats when a small number of W-256 cells were injected interdigitally into the acute inflammatory swellings produced by the local injection of paws with LH or CS.Acute inflammatory reactions were induced in rats by the intravenous injection of cellulose sulphate (CS) or an extract of normal rat lung homogenate (LH), or by intraperitoneal injections of Compound 48/80. These treatments greatly increased survival and clonogenic growth in the lungs of rats of intravenously injected allogeneic W-256 and Y-P388 tumour cells. Increase in the dose of intravenously injected CS caused a logarithmic increase in colony forming efficiency (CFE) of tumour cells in the lungs. CFE was not stimulated by the intravenous injection of rats with pharmacological mediators of inflammation (histamine, 5-hydroxytryptamine, bradykinin and prostaglandins PGECS is a “synthetic heparin” which causes marked prolongation of blood clotting time and also increases fibrinolytic activity of the blood. Anticoagulant treatment of rats with heparin did not affect CFE. Thus, there was no direct correlation between blood clotting time and CFE of blood borne tumour cells in the rat.The mechanisms which may be responsible for the nonspecific growth promoting effects of inflammatory reactions induced by various types of tissue injury on tumour induction and growth are discussed.


ZD6126 could be stably encapsulated, and liposomes displayed minimal leakage in vitro (<10% in 3 weeks). In vivo, upon intravenous injection, free ZD6126 was rapidly converted into ZD6126 phenol, which was cleared from the circulation within minutes. In contrast, ZD6126 encapsulated into either RGD-targeted or PEG liposomes showed prolonged blood circulation times (t1/2=10 h), and ZD6126 phenol exposure was also prolonged (t1/2=8 h). Both liposomal formulations displayed tumour accumulation plus hepatosplenic uptake by local macrophages. The altered pharmacokinetics and tissue distribution profiles of both liposomal ZD6126 formulations resulted both in single-dose and multiple-dose regimes, in improved therapeutic efficacy in established murine B16.F10 melanomas compared with free ZD6126. The passively and actively targeted liposomes showed equal antitumour efficacy, indicating that delivery of ZD6126 to the tumour tissue may suffice to disrupt tumour blood vessels without the need for specific targeting to the tumour endothelium.Vascular disrupting agents (VDAs) are able to affect selectively tumour endothelial cell morphology resulting in vessel occlusion and widespread tumour cell necrosis. However, single-agent antitumour activity of VDAs is typically limited, as tumour regrowth occurs rapidly following drug treatment. To improve the therapeutic effectiveness of VDAs, we investigated liposomal targeting using ZD6126 as a model VDA. ZD6126 is a phosphate-prodrug of the tubulin-binding vascular disrupting agent ZD6126 phenol. ZD6126 was encapsulated into long circulating PEG-liposomes for passive targeting and PEG-liposomes conjugated with peptide ligands containing the RGD-motif for active targeting to  Blood vessel development is important for tumour progression. Therapies that specifically focus on targeting tumour vasculature have been extensively explored since the early 1970s . VasculaTumour vessels are different from quiescent endothelium at various levels. For example, tumour vasculature generally lacks the structural support of mature vessels, is more permeable, and also expresses distinct antigens . FurtherA number of second-generation compounds have been extensively studied in preclinical settings including ZD6126, Combretastatin A4 phosphate, AVE8062, ABT-751, and OXi4503. Pronounced antitumour effects were seen in a wide variety of preclinical tumour models . DespiteIn clinical trials, this class of compounds has shown marked reductions in tumour blood flow, although single-agent antitumour efficacy has been very limited, as predicted from the preclinical models . LiposomN-acetylcolchinol), which is formed after rapid hydrolysis of the prodrug by phosphatases present in circulating blood.In this study, we investigated whether the therapeutic effectiveness of ZD6126 could be increased by targeting the agent to the tumour tissue. ZD6126 is a water-soluble phosphate prodrug of the tubulin-binding agent ZD6126-phenol  peptides to the surface of the PEG liposomes, which specifically bind to αv-integrins that are overexpressed on the surface of angiogenic endothelial cells (Intravenously (i.v.) injected ZD6126 has a very fast elimination rate from the blood in both rats and humans with μM ml−1. After dissolving the lipids in chloroform:methanol (2 : 1 vol:vol), a lipid film was made under reduced pressure on a rotary evaporator and dried under a stream of nitrogen. Liposomes were formed by addition of an aqueous solution (40–80 mg ml−1) of ZD6126. Approximately 150-nm-sized liposomes (Polydispersity index <0.2) were obtained by repeated extrusion. Encapsulated ZD6126 concentration was determined by high-pressure liquid chromatography  using a gradient of 80% 1000 : 1 water:trifluoroacetic acid (solvent A)/20% 1000 : 1 acetonitrile:trifluoroacetic acid (solvent B) to 10% solvent A and 90% solvent B over 9 min, as a mobile phase.Long circulating liposomes were prepared as described previously  per μM total lipid was added after deacetylation of the peptide in 0.5 M hydroxylamine solution and incubated for 1 h at room temperature. Unloaded ZD6126 and unbound RGD were separated from the liposomes by PD-10 column . To obtain 10 mg ml−1 ZD6126 liposomal formulations, liposomes were concentrated by ultracentrifugation for 1 h at 60 000 r.p.m. .Cyclic RGD (ARG-GLY-ASP) peptide coupling was performed as described previously  and heat-inactivated FBS-supplemented Dulbecco's modified Eagle's medium, respectively. Cells were seeded (1 × 104 cells per well) in 96-well plates 24 h before samples were added. ZD6126 formulations (0.01–100 μM) were incubated with the cells for 24 h and finally an XTT cell viability assay was performed , B16F10 murine melanoma cells and J774A.1 murine tumour-derived macrophages were grown at 37 °C and 5% CO−1) was administered i.v. in the tail vein to C57Bl/6 mice (n=3 per group). Blood samples were collected by cardiac puncture at various time intervals (5 min to 48 h) after dosing. Plasma levels of ZD6126 and ZD6126 phenol were determined by tandem mass spectrometry (PE Sciex API 3000 spectrophotometer). The limits of quantification were 10 ng ml−1 for ZD6126 and ZD6126 phenol.PEG-liposomal (RGD-coupled) and free ZD6126 (20 mg kg−1)-treated B16F10 mice were killed at several time points and tumours (>100 mm3), livers and spleens were excised, snap-frozen in liquid nitrogen and stored at −80 °C. After acetone fixation, 5 μm sections were stained for the RGD peptide. Staining of RGD was performed as described previously  1 : 40 (in PBS+BSA 1%) was used as primary antibody after serum blocking with normal rabbit serum (1 : 20 in TBS-Tween 20). Subsequently, staining of endogenous peroxidase and biotin blocking  was performed followed by incubation with biotinylated rabbit anti-rat IgG  and horseradish peroxidase-streptavidin . For both staining procedures, 3-amino-9-ethylcarbazole  was used as a substrate and hematoxylin (Sigma-Aldrich) for counterstaining. Slides were mounted in Kaiser's glycerol-gelatin medium  and visualised by light microscopy (Nikon TE2000-U).ZD6126-loaded RGD-PEG-liposome (20 mg kgMicroscopic fields (200 × magnified) per sample were analysed for stained regions (sum of substrate-stained pixels) with computerised image analysis programme (Image-Pro® plus 4.5 for windows). Computer software was used to quantify total substrate (red) stained areas. Finally, data were corrected for tissue weight to calculate total staining per gram of tissue.n=10 per group, average tumour size=255±143 mm3) were treated with free ZD6126 (100 mg kg−1), RGD-liposomal ZD6126 (100 mg kg−1) or liposomal PBS control and tumours were excised 24 h afterwards. Subsequent to fixation in 10% buffered formalin and standard processing to paraffin wax blocks, sections (5 μm) were prepared and stained with H&E. The level of necrosis was scored blinded to treatment details by a pathologist. Scores can vary between 1 and 10, where 1 specifies 0–10% necrosis and 10 denotes >90–100% necrosis , PEG-liposomal ZD6126 (100 mg kg−1) or RGD-PEG-liposomal ZD6126 (100 mg/kg) was administered i.v. in the tail vein. Tumour size was measured every second day, using digital calipers. For the multiple 100 mg kg−1 dose study, mice were also treated for the first time at day 11 followed by two injections at 4-day intervals (days 15 and 19). Tumour volumes were calculated using the following equation: V=(A × 0.52) × B2, where A is the largest and B is the smallest superficial diameter. Mice were killed when tumour reached a volume of 2000 mm3, if tumour had broken open through the skin or when animals appeared moribund.C57Bl/6J mice were injected subcutaneously in the right flank with 1 × 10All animal studies were validated and performed according to Utrecht University ethical guidelines, in line with national laboratory animal regulations.For the necrosis scores, Mann–Whitney rank-sum test was used, and for analysis of the survival curves the Log-rank test was applied.μm and a polydispersity index below 0.2, indicating limited variation in particle size. Hydration of 100 mM total lipid with 40 mg ml−1 ZD6126 yielded an average concentration of 7.5±2.5 mg ZD6126 per ml of liposome suspension. ZD6126-loaded liposomes showed less than 10% leakage in PBS, at 37 and 4 °C over 3 weeks. Particle size was maintained over this time period.ZD6126-loaded PEG liposomes or RGD-PEG-liposomes were prepared by the lipid-film hydration method. The amount of encapsulated drug increased proportionally with the total amount of ZD6126 added. A positive correlation between liposome diameter and amount of encapsulated drug was observed, indicating that the ZD6126 is primarily present in the aqueous interior of the liposomes. The liposomes that were used in these studies had a mean diameter of 0.15 μM did not induce cytotoxicity  was rapidly (t1/2=0.04 h) converted to ZD6126-phenol and quickly (t1/2=0.08 h) cleared from the blood  blinded to treatment. Vehicle-treated controls showed some necrosis. In this tumour model, free ZD6126 (100 mg kg−1) did not show a significant difference in the degree of tumour necrosis compared with vehicle-treated controls (−1 ZD6126)-treated tumours showed a significantly increased tumour necrosis score compared with control. As a control, 200 mg kg−1 free ZD6126 was administered and showed >90% necrosis 24 h after injection  i.v. with 100 mg kgcontrols . Similarcontrols . Howevercontrols . HoweverIn vitro cytotoxicity studies confirmed limited leakage from the liposomes. Human umbilical vein endothelial cells, murine melanoma cells (B16.F10), as well as macrophages (J774A.1) all showed a 10-fold decreased cytotoxicity for the liposome-encapsulated ZD6126 as compared with the free drug, which is consistent with <10% drug release in buffer. This conclusion is supported by previous studies that showed limited cellular interaction of PEG liposomes (in vivo in mice, as both the parent drug ZD6126 and its active moiety ZD6126 phenol have a short half-life. However, as PEG liposomes tend to accumulate in macrophages, sustained high levels of ZD6126 may be reached in this cell type after liposomal administration of ZD6126.Vascular disrupting agents have been widely explored as antitumour agents in preclinical and clinical studies. In this study, we have shown substantial improvement in the antitumour activity of the VDA ZD6126 by liposomal encapsulation. Water-soluble ZD6126 was stably associated with the liposomes. −1 dosing schedule. However, 200 mg kg−1 free ZD6126 showed >90% necrosis 24 h after injection (Intravenously administered free ZD6126 and its dephosphorylated active form ZD6126 phenol were cleared from the circulation within 15 min . These onjection , indicatFor both RGD-PEG and PEG liposomes, tumour growth delay persisted much longer than with free ZD6126, most likely as a result of increased local and systemic drug exposure. This is in contrast to a single earlier report, using actively targeted RGD-PEG liposomes to deliver the VDA combretastatin A4 (There was no apparent difference between the two liposome formulations, indicating that the association of RGD-PEG liposomes with tumour endothelium does not improve therapeutic efficacy compared with PEG liposomes, which do not display a specific endothelial cell interaction. To intensify the antitumour effects, a multiple dosing schedule was evaluated. Multiple doses of free drug prolonged the antitumour effects. Also for the liposomal formulations, multiple injections significantly prolonged tumour growth delay; however, after the third injection, some mice became moribund. It is at present unclear why the liposomal ZD6126 was not tolerated after the third injection, as toxicological studies have not been performed, although decreased tolerability could result from an altered tissue distribution profile and/or increased drug exposure. The time interval between doses of liposomal ZD6126 to optimise antitumour effects and minimise toxicity warrants further investigation.In conclusion, liposomal encapsulation of ZD6126 improves monotherapeutic antitumour activity compared with a similar dose of free ZD6126. The prolonged antitumour effects observed for the liposomal formulations could also be beneficial for combination therapy strategies, extending the time to attack remaining viable tumour cells. RGD-PEG liposomes, targeted to angiogenic endothelial cells, proved to be no more efficacious than passively targeted PEG liposomes, indicating that active targeting and cell-specific delivery of VDAs do not offer an advantage over target tissue delivery in this tumour model. After phosphatase cleavage, ZD6126 phenol can traverse cellular membranes and bind to its intracellular target, tubulin, which could reduce the importance of target cell-specific delivery. This would mean that locally accumulated liposomes serve as a depot for ZD6126, increasing tumour site drug exposure compared with the rapidly cleared free ZD6126. This increase in tumour drug exposure could also induce alternative mechanisms of action for ZD6126, including tumour cell and tumour-associated macrophage toxicity, which could contribute to overall antitumour effects.


The most common cause of death of cancer sufferers is through the occurrence of metastases. The metastatic behaviour of tumour cells is regulated by extracellular growth factors such as hepatocyte growth factor (HGF), a ligand for the c-Met receptor tyrosine kinase, and aberrant expression/activation of the c-Met receptor is closely associated with metastatic progression. Nk4 32b) is a competitive antagonist of the HGF c-Met system and inhibits c-Met signalling and tumour metastasis. Nk4 has an additional anti-angiogenic activity independent of its HGF-antagonist function. Angiogenesis-inhibitory as well as cancer-specific apoptosis inducing effects make the Nk4 sequence an attractive candidate for gene therapy of cancer. This study investigates the inhibition of tumour metasasis by gene therapy mediated production of Nk4 by the primary tumour. Optimal delivery of anti-cancer genes is vital in order to achieve the highest therapeutic responses. Non-viral plasmid delivery methods have the advantage of safety and ease of production, providing immediate transgene expression, albeit short-lived in most tumours. Sustained presence of anti-angiogenic molecules is preferable with anti-angiogenic therapies, and the long-term expression mediated by Adeno-associated Virus (AAV) might represent a more appropriate delivery in this respect. However, the incubation time required by AAV vectors to reach appropriate gene expression levels hampers efficacy in many fast-growing murine tumour models. Here, we describe murine trials assessing the effects of Nk4 on the spontaneously metastatic Lewis Lung Carcinoma (LLC) model when delivered to primary tumour via plasmid lipofection or AAV2 vector. Intratumoural AAV-Nk4 administration produced the highest therapeutic response with significant reduction in both primary tumour growth and incidence of lung metastases. Plasmid-mediated therapy also significantly reduced metastatic growth, but with moderate reduction in primary subcutaneous tumour growth. Overall, this study demonstrates the potential for Nk4 gene therapy of metastatic tumours, when delivered by AAV or non-viral methods. HGF is a heterodimeric molecule and functions include mitogenic, motogenic, morphogenic and anti-apoptotic activities ,2. HGF pin vitro with pEGFP-F delivery as assessed by fluorescent microscopy (data not shown), and in vivo (figure in vitro by RT-PCR (figure in vivo experiments were approved by the ethics committee of University College Cork. Subcutaneous (s.c.) LLC tumours were induced in 6–8 week old female C57 mice obtained from Harlan Laboratories  using 2 × 105 cells, suspended in 200 μl serum free Dulbecco Modified Eagle Medium, DMEM,  injected subcutaneously into the flank. When the tumours reached an average volume of 0.1 cm3, they were intratumourally (i.t.) administered 75 μl plasmid/lipofectamine2000 mix containing 25 μg DNA corresponding to Nk4-coding or Backbone (BB) plasmid, or received no treatment (n = 9). The firefly luciferase coding plasmid pCMVluc  was also administered to a group to validate transfection (n = 3) and IVIS imaged at 24 h. 1.64 × 10-8 p/sec/cm2/sr/plasmid copy was observed confirming the transfectability of LLC tumours by this method ∏/6. At each time point, a two-sampled t-test was used to compare mean tumour volume within each treatment group. Microsoft Excel (Microsoft) was used to manage and analyze data. Statistical significance was defined at the standard 5% level. Figure Tumour growth was monitored by alternate day measurements in two dimensions using a Verniers callipers. Tumour volume was calculated according to the formula V = . AAV2 vector particles were prepared using the Stratagene AAV Helper Free System , and concentrated using the Virakit system . Cells transduced with AAV-LacZ (Stratagene) particles were assessed for β-Galactosidase activity microscopically to determine the titre of the particle stocks, in parallel with AAV-Nk4 and AAV-BB. Nk4 expression was validated by reverse transcription PCR (RT-PCR) (Omniscript RT kit (Qiagen)) using primers forward CCTCTCTGATGACATGAAGAAG and reverse TGTCACAAAAGCTCTCCCC.While both plasmid and adenoviral vectors have been utilised for Nk4 gene therapy of cancer -11, the 3, i.t. administered 107 particles/40 μl AAV-Nk4, AAV-BB or PBS (n = 9). Tumour volumes were measured at regular intervals and 3 mice of each group were culled at 2 time points during the trial for analysis of lung metastases. The effects of AAV particles on tumour growth are detailed in figure Subcutaneous LLC tumours were induced in C57 mice and at an average volume of 0.1 cmPulmonary metastatic burden was assessed by visual counts at day 21 and 26. Mice in the control groups (BB and Untreated) showed more metastatic burden on both time points than the AAV-Nk4 treated group, and the BB group displayed increased  metastatic burden over the untreated group (data not shown). Combined Day 26 and Day 21 measurements are shown in figures 3) was day 7. Others have addressed the limitation associated with AAV delayed expression by the use of self-complementary AAV [in vitro [-3 p/sec/cm2/sr/AAVparticle vs 10-8 p/sec/cm2/sr/plasmid copy). The significant differences in effects on tumours between the Nk4 containing and Nk4-free controls, coupled with demonstration of in vivo reporter gene expression in LLC tumours, as well as in vitro Nk4 expression data, indicate that Nk4 sequences were responsible for the observed effects on tumour growth.It has previously been reported that by day 6 post tumour inoculation, 100% LLC mice have already developed metastatic disease . In our tary AAV . It is pin vitro . We obseDuration of gene expression is an important factor to be addressed in such gene therapies. It has previously been reported that slow release of NK4 plasmid DNA from cationised gelatin increases efficacy of Nk4 plasmid therapy . While wThe nature of our LLC model meant that it was not possible to generate survival curves based on death due to metastatic disease, as trials had to be stopped at or prior to 26 days post tumour inoculation due to primary tumour size. Plasmid experiments were ceased at day 21, due to early ulceration of tumours at subsequent times in plasmid administered groups, possibly related to toxicity of lipofectamine. No such ulceration was observed in AAV administered tumours up to day 26. A tumour model permitting longer-term study of this therapy would yield further information. Given the distance from clinical reality of fast-growing murine tumour models, anti-metastatic therapy as described here may yet prove a powerful therapeutic strategy in humans, especially if applied earlier in tumour progression.The authors declare that they have no competing interests.in vitro and in vivo experiments, and contributed to drafting the manuscript. SAC and SR aided in generation of AAV vector particles and in vivo trials. JPvP designed and aided in cloning of AAV plasmids. MM constructed AAVCMVluc. GCO'S, DMO'H and MT were the coordinators of the project. MT designed the studies and drafted the manuscript. All authors read and approved the final manuscript.AB performed the


A novel 111In ligand , termed 9N3, was covalently attached to chimeric B72.3, labelled with 111In and compared with 111In-labelled chimeric B72.3 diethylenetriaminepentaacetic acid (DTPA) cyclic anhydride conjugate (cDTPA) and a C-linked derivative of DTPA (CT-DTPA) in athymic mice bearing human colon carcinoma xenografts. Significant differences in biodistribution were observed between 9N3 and cDTPA conjugates especially in the tumour uptake and blood, liver, femur and colon levels at 24, 48 and 144 h. Significantly higher tumour uptake was observed for 111In-cB72.3-9N3 compared with 111In-cB72.3-cDTPA at all time points. Radiolocalisation (RI) indices increased with time for the 9N3 conjugate but remained constant for the cDTPA conjugate. The biodistribution of 111In-labelled cB72.3-CT-DTPA was similar to that of 111In-labelled cB72.3-9N3 except for elevated kidney levels. A 12N4 macrocycle  was also tested for its ability to chelate 111In and its biodistribution examined. Labelled conjugates with this macrocycle were more difficult to prepare in a stable form but gave a very similar biodistribution to the 9N3 macrocycle conjugate. Macrocycle-antibody conjugates of this type offer considerable promise for tumour imaging in patients.


Fibrinogen radioiodinated by the iodine monochloride method was tested as a tumour radiodiagnostic agent in mice. The I-fibrinogen cleared from the blood of tumour-bearing mice more rapidly than from that of normal mice, but it cleared from the whole body more slowly, suggesting it accumulated in a substantial tumour-related compartment in the abnormal mice. The tumour concentration steadily increased for 4 h after injection, at which time it reached a peak concentration of 11-4% of the injected dose/g. This concentration was higher than the peak concentration for Ga-citrate (not reached until 24 h) or any other oncophilic radiopharmaceutical tested in this tumour model. The early accumulation is consistent with the use of 123I as a tracer label for fibrinogen. A combination of the large tumour concentration of I-fibrinogen, an increased catabolic rate induced by chemical modification, and the exceptional nuclear properties of 123I for scintigraphic imaging, could lead to a very useful radiodiagnostic procedure for cancer.


P = 0.0005), and increasing median survival from 19 to 30 days (P = 0.0001). In addition, carcinoembryonic antigen (CEA) levels were measured in serum samples from animals sacrificed at regular intervals, and correlated with excised tumour weight. It was shown that the natural log of the CEA concentration was linearly related to the natural log of the tumour weight and that treatment was not a significant factor in this relationship (P = 0.7). In conclusion, circulating CEA levels were not directly affected by marimastat, but did reflect tumour size. These results support the use of cancer antigens as markers of biological activity in early phase trials of non-cytotoxic anticancer agents. © 1999 Cancer Research CampaignInhibition of matrix metalloproteinases (MMPs) is an attractive approach to adjuvant therapy in the treatment of cancer. Marimastat is the first orally administered, synthetic MMP inhibitor to be evaluated, in this capacity, in the clinic. Measurement of the rate of change of circulating tumour antigens was used for evaluating biological activity and defining optimum dosage in the early clinical trials of marimastat. Although tumour antigen levels have been used in the clinical management of cancer for many years, they have not been validated as markers of disease progression. In order to investigate the relationship between the effects of marimastat on tumour growth and circulating tumour antigen levels, mice bearing the human gastric tumour, MGLVA1, were treated with marimastat. The MMP inhibitor exerted a significant therapeutic effect, reducing tumour growth rate by 48% (


A series of 46 primary bronchogenic carcinomas for which thymidine labelling index (%TLI)  and tumour doubling time (DTact) (in 13 cases) had previously been measured were followed up for 5 years and these data compared with length of post operative survival, tumour volume at operation and pathological staging. We found no correlation between reduced survival and higher tumour %TLI, indeed the reverse may be true. Larger tumours tended to have higher labelling indices considering either primary tumour volume or 'T'-category. Five year survivors had smaller tumours, tended to have T1 tumours and Stage I disease but did not have significantly lower tumour %TLIs. No relationship was found between DTact and any other parameter.


The tumour response was essentially the same for both the single and fractionated dose schedules.The effects of one large single dose of 5-fluorouracil (FU) have been compared to the same amount given in divided doses daily over a 3- or 5-day period. Comparison of the effects of single There were marked increases in animal mortality from drug toxicity following fractionated doses of FU compared to one large single dose. Mortality in animals with Tumour 3924A increased from 10% following one large single dose to 60% for animals given daily fractionated doses for 3 days, and 80% for animals given daily fractionated doses for 5 days. Total marrow reserve was measured by the total DNA content of tibial marrow. The nadir of 6 days for loss of total tibial marrow DNA following one large dose of FU was increased to 9 days for both fractionated schedules of FU. The 3-day delay in recovery of the marrow prevented recovery within the time frame necessary for animal survival. The inference from these experimental cancer-treatment studies is that daily fractions of chemotherapeutic agents such as FU result in increased morbidity and mortality, without benefit in the control of the solid tumour. The results question the advisability of the clinical practice of initially giving small daily “loading doses” of proliferation-dependent agents such as FU.These results emphasize the need for more precise information on the temporal relationship between the response and recovery of the host and the response and recovery of the solid tumour. They also emphasize the need for a better clinical understanding of the time sequence of solid-tumour recovery in relation to the time sequence of marrow recovery.


In a study of DNAs from 100 breast cancer patients and 100 controls, there were no differences in the frequencies of common or rare alleles at the Harvey ras (c-Ha-ras) locus on chromosome 11. However, one Ha-ras allele was deleted from the tumour DNA in 14 of 65 informative patients. Loss of a Ha-ras allele correlates with paucity of oestrogen receptor protein and with increased tumour size at presentation, but is not associated with microscopic evidence of lymph node invasion. The findings on Ha-ras and other informative loci are consistent with the possibility that a tumour suppressor gene involved in the early stages of breast cancer is located on the short arm of chromosome 11.


The selective recognition of TES-23 to TECs was competitively blocked by preadministration of unlabelled TES-23 in vivo. Furthermore, immunostaining of human tissue sections showed specific binding of TES-23 on endothelium in oesophagus cancers. These results indicate that tumour vascular endothelial cells express common antigen in different tumour types of various animal species. In order to clarify the efficacy of TES-23 as a drug carrier, an immunoconjugate, composed of TES-23 and neocarzinostatin, was tested for its anti-tumour effect in rats bearing KMT-17 fibrosarcomas. The immunoconjugate (TES-23-NCS) caused marked regression of the tumour, accompanied by haemorrhagic necrosis. Thus, from a clinical view, TES-23 would be a novel drug carrier because of its high specificity to tumour vascular endothelium and its application to many types of cancer. © 1999 Cancer Research CampaignThe tissue distribution of anti-tumour vascular endothelium monoclonal antibody (TES-23) produced by immunizing with plasma membrane vesicles from isolated rat tumour-derived endothelial cells (TECs) was assessed in various tumour-bearing animals. Radiolabelled TES-23 dramatically accumulated in KMT-17 fibrosarcoma, the source of isolated TECs after intravenous injection. In Meth-A fibrosarcoma, Colon-26 adenocarcinoma in BALB/c mice and HT-1080 human tumour tissue in nude mice, radioactivities of


The biodistribution of no-carrier-added (n.c.a.) meta-[131I]iodobenzylguanidine ([131I]MIBG) and that prepared by the standard isotopic exchange method were compared in athymic mice bearing SK-N-SH human neuroblastoma xenografts. No advantage in tumour uptake was observed for the n.c.a. preparation. BALB/c nu/nu mice exhibited lower uptake in highly innervated normal tissues  than normal BALB/c mice. In another experiment, the distribution of n.c.a. [131I]MIBG in the absence or presence (3-9 micrograms) of MIBG carrier was determined. At both 4 h and 24 h, the heart uptake was reduced by a factor of 1.5 even at a dose of 3 micrograms MIBG. Tumour uptake was not significantly altered by various amounts of unlabelled MIBG at either time point.


The tumour specific transplantation antigen (TSTA) from a chemically induced rat sarcoma has been isolated as an electrophoretically homogeneous soluble material by affinity chromatography using a Sepharose bound antibody raised to the tumour in syngeneic rats. The TSTA is specific for the particular tumour used (the MC-1 sarcoma) and does not cross-react with material extracted from other rat sarcomata. In addition, a material with different physicochemical properties which cross-reacted with different sarcomata was also eluted from the antibody column and this may be the previously identified onco-embryonic antigen (OEA1) which is immunogenic in the syngeneic host.125I was used in a radioimmunoassay which detected soluble TSTA in rats bearing a MC-1 sarcoma. The assay shows that tumour transplantation is associated with a persisting release of soluble antigen into the circulation. This antigenic burden is present continuously and renewed as long as the tumour mass exists.The purified TSTA labelled with


F), and blood volume (V) were modelled. Imaging was combined with pO2 measurements, analysis of microvessel density, microvessel diameter, microvessel fractal dimension, and expression of vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1 α (HIF-1α), Bax, and Bcl-2. We found that RT significantly reduced PS and V in control rats, but not in rhEPO-treated rats, whereas F was unaffected by RT. Oxygenation was significantly better in rhEPO-treated animals, and RT induced a heterogeneous reoxygenation in both groups. Microvessel diameter was significantly larger in rhEPO animals, whereas VEGF expression was significantly lower in the rhEPO group. No differences were observed in HIF-1α, Bax, or Bcl-2 expression. We conclude that rhEPO results in spatially heterogeneous modulation of RT effects on tumour microvessels. Direct effects of rhEPO on neoplastic endothelium are likely to explain these findings in addition to indirect effects induced by increased oxygenation.Recent data suggest that recombinant human erythropoietin (rhEPO) modulates tumour growth and therapy response. The purpose of the present study was to examine the modulation of radiotherapy (RT) effects on tumour microvessels by rhEPO in a rat colorectal cancer model. Before and after 5 × 5 Gy of RT, dynamic contrast-enhanced -magnetic resonance imaging was performed and endothelial permeability surface product (PS), plasma flow ( Anaemia commonly occurs in colorectal cancer patients especially if they are treated with neoadjuvant radiotherapy (RT) or chemotherapy. Anaemia not only adversely affects the clinical condition of these patients, but also contributes to the development of tumour hypoxia, recognised as a major negative determinant of sensitivity to RT, chemoradiotherapy, and certain chemotherapeutic agents  increases haemoglobin levels and improves quality of life in patients with cancer-related anaemia . PreclinThe importance of tumour oxygenation for RT response is well established, and there has been considerable interest in modulating tumour oxygenation and RT response by rhEPO administration. Experimentally, exogenous rhEPO has been shown to improve or restore radioresponsiveness in both anaemic and nonanaemic animals  expression and microvessel morphology  with a macromolecular contrast agent (CA) to demonstrate a significantly decreased neovascular leakage after fractionated RT in a rat colorectal cancer model   additionally supplemented with 10% fetal calf serum, 4 mM L-glutamine, 50 U ml−1 penicillin, and 50 μg ml−1 streptomycin at 37°C in a humidified atmosphere with 5% CO2 in air. The cells were transferred at 95% confluency. Two million cells suspended in 0.2 ml of saline were injected subcutaneously (s.c.) in the proximal hind leg. Tumours reached a size of 0.5–1 cm after a period of 4 weeks. Once a tumour growth of minimally 8 mm diameter was observed, a jugular vein catheter was inserted and tunnelled to the interscapular region. To maintain catheter patency, continuous infusion at 0.5 ml saline per hour was administered with a cage-mounted swivel and a flexible metal tether system  allowing the animal full mobility.Male Wag/Rij rats were bought from Harlan . The CC531 cell line is a 1,2-dimethylhydrazine-induced, moderately differentiated, and weakly immunogenic colon adenocarcinoma, syngeneic with WAG/Rij rats. This cell line is well studied and has been proven to provide a tumour–host model similar to human colorectal carcinogenesis (n=11) and an rhEPO group (n=15) receiving rhEPO  at a dose of 3 × 0.1 ml (286 IU) s.c. per week. The dosage was based on a dose-finding study during which five or eight rhEPO administrations weekly resulted in an excessive haematocrit rise and important mortality (data not shown).Recombinant human EPO has been shown to bind to the rodent EPO receptor ; first DCE-MRI, oxygenation and flow measurement (day 8); fractionated RT 5 × 5 Gy (day 13–17); second DCE-MRI, oxygenation and flow measurement, and killing by anaesthesia overdose and excision of tumours for histology .Rats were not sedated and the tumour-bearing hind leg was immobilised using a Plexiglass holder, as described previously . Animals were sedated with 0.2–0.4 ml of medetomidine . Imaging comprised a single axial slice that was positioned through both lower limbs and the centre of the tumour. Before the contrast series, T1 zero time maps were constructed from two spin echo sequences with different repetition times . Details of this sequence were as follows: slice thickness 3 mm, field of view (FOV) 140 × 88, matrix size 256 × 160, echo time (TE) 20 ms, and flip angle 90°. Dynamic imaging was performed with a 4-antenna wrist coil (diameter 10 cm) using an inversion recovery TurboFLASH sequence. Details of the pulse sequence were as follows: temporal resolution 1.1 s (i.e. TR 1100 ms), FOV 140 × 88, matrix size 256 × 160, slice thickness 5 mm, TE 4.08 ms, inversion time 560 ms, and flip angle 12°. A bolus of 0.3–0.4 ml of P792 was manually injected as fast as possible (approximately 1 ml s−1) through a central venous line after the fourth scan. A total of 500 images was obtained for a total scan time of 550 s.TVp) and fractional extracellular volume (Ve) reached by the CA equals 100%, that is, Vp+Ve=1.Pixel-by-pixel pharmacokinetic modelling of DCE-MRI data was performed with the research mode of a dedicated software package . Extraction of both microvascular permeability and flow data was based on the tissue homogeneity (TH) model of capillary exchange originally described by When the assumption is made that changes in EES concentration per unit time are negligible compared with changes in plasma concentration, an adiabatic approximation to the TH model can be derived, which has been used in modelling of DCE-MRI CA kinetics applied to tumour microvasculature (Ce (mM) is the CA concentration in the EES, Fp (unitless) is the plasma flow, R denotes the residue function, and ⊗ is the convolution operator.The adiabatic approximation to tissue homogeneity (AATH) model is given by superposition of the arterial input function (AIF) with a time-varying residue function: t<τ, with tau as the transit time through a capillary) and a tissue phase (t>τ). Depending on the time interval, the residue function will encompass a vascular and a tissue component: E the extraction ratio from plasma space to EES, Ktrans the endothelial transfer constant (min−1), ve the fraction of the EES available as leakage space (unitless), and τ is the mean capillary transit time (s).The time course of CA arrival is divided in a vascular phase . After each micromanipulator movement, measurements were started as soon as a stable reading was obtained. Tissue pO2 was sampled every 2 s. Over this 4 mm trajectory, oxygenation and LDF values were recorded separately for the tumour core  and peripheral angiogenic rim (outer 1 mm). Tissue pO2 was expressed in mmHg, whereas LDF was expressed in arbitrary units (a.u.).Tissue oxygenation and laser Doppler flow (LDF) were measured with a fibreoptic probe combining fluorescence quenching with laser Doppler flowmetry  (α (HIF-1α) . Paraffin-embedded sections were rehydrated by serial immersion in xylene and ethanol. After rinsing, the endogenous peroxidase was blocked with 0.3% hydrogen peroxide. The sections were subsequently incubated with a biotinylated secondary antibody, followed by incubation with a streptavidin–peroxidase complex . The colour reaction was developed using 3-amino-9-ethylcarbazole substrate (Dako) as chromogen. Finally, the sections were counterstained with haematoxylin. Semiquantitative scoring was based on a method modified after Paraffin-embedded tissue samples were used for immunohistochemistry with the following antibodies: anti-EPO receptor (M-20) , anti-Bcl-2 , anti-Bax , anti-VEGF , and anti-hypoxia-inducible factor-1 μm frozen slices with anti-CD31 antibodies , the entire tumour section was scanned at low-power  to identify ‘hot spots’, which are the areas of highest neovascularisation. Individual microvessels were then counted under higher power  to obtain a vessel count in a defined area, and the average vessel count in three hot spots was taken as the MVD.Microvascular density (MVD) was determined with a method modified after http://rsb.info.nih.gov/ij).Microvascular diameter was measured on digitised CD31-stained slices . From each rat, five different zones were analysed and the largest diameter was measured from all visible microvessels using NIH ImageJ software  and analysed with ImageJ software. By applying a colour threshold, non-CD31-stained pixels were removed from the image. The microvessel fractal dimension (MFD) was calculated using the box-counting method. The image is divided into increasingly smaller boxes, and after each step the number of nonempty boxes is counted.ε the length of a box side and N(ε) the smallest number of boxes required to contain all CD31-stained pixels. In practice, the limit of a box sized 0 cannot be applied and therefore the MFD was calculated as the slope of the curve fitted after plotting log N(ε) vs log (1/ε). Calculations were performed with the fractal dimension plugin available in the ImageJ environment.The fractal dimension is calculated as t-test or, when data distribution was non-Gaussian, with the nonparametric Mann–Whitney U-test, whereas differences between fractions were evaluated with the χ2 or Fisher's exact test. Statistical significance was assumed when P⩽0.05. All calculations and plotting were performed with SigmaStat software .Data are expressed as mean±standard error (s.e.) of the mean, unless stated otherwise. Differences between two groups of continuous data were analysed with the Student's All tumours showed expression of the EPO-R on neoplastic cells and neoplastic endothelium . There wP<0.001, Student's t-test). In the control group, haematocrit values remained unchanged.Haematocrit values were tested before starting the experiment and on day 8 (immediately before the first MR imaging and oxygenation measurements). In rhEPO-treated rats, mean haematocrit showed a 25% increase from 50.7±0.4% before therapy to 62.6±0.4% on day 8 . After RT, tumour volume was 0.99±0.2 cm3 in the control group and 0.99±0.16 cm3 in the rhEPO group .The present experiment was intended to detect early microvascular changes after fractionated RT and not to study the effects of rhEPO on tumour growth or modulation of RT effects on tumour growth. Administration of rhEPO was therefore started after a tumour had developed. Tumour volume before RT was 1±0.2 cmFp, permeability surface product PS, and fractional plasma volume Vp.Pixel-by-pixel kinetic modelling encompassing both the tumour core and rim was performed with estimation of three microvascular parameters: plasma flow P<0.001, Student's t-test). No significant effect of RT was noted in the tumour rim of control animals. In rhEPO-treated rats, microvascular flow in the tumour core was also significantly lower after RT  but unaffected in the tumour rim. The magnitude of the RT effect on tumour core flow did not differ significantly between control and rhEPO-treated animals .Results of DCE-MRI microvascular data modelling are shown in P<0.001, Student's t-test) but not in the tumour core. In rhEPO-treated animals, however, PS was not affected by RT.In control animals, the microvessel PS was significantly lowered by RT in the tumour rim . In rhEPO-treated animals, however, no significant changes in plasma volume were noted after RT.Fractional plasma volume was significantly lower in both the tumour core and rim in control animals after RT  but not in the tumour rim . In the rhEPO group, on the contrary, no significant difference in oxygenation was observed in the tumour core , whereas a significant increase in pO2 was observed in the tumour rim . Both before and after RT, pO2 values were significantly higher in rhEPO-treated rats in both regions of the tumour (data not shown).Oxygenation and LDF measurements were performed before and 5 days after completion of fractionated RT. Mean pOP=0.023, Student's t-test), but not in the tumour rim. In the rhEPO group, however, RT did not influence LDF in either tumour zone. Before RT, LDF measured in the tumour core was significantly lower in rhEPO-treated animals  compared with controls, whereas no significant difference was present in the tumour rim. After RT, LDF values in both tumour core and rim were not significantly different between controls and rhEPO-treated animals (data not shown).Mean LDF values (a.u.) are illustrated in P=0.35, Student's t-test). Within each group, MVD was significantly lower in the tumour core compared with the tumour rim .Mean microvascular density was 12.5±1.2 in the control group and 14.3±1.4 in the rhEPO group . In the tumour rim, however, MFD did not differ between controls and rhEPO-treated animals , Microvessel morphology data are illustrated in μm) in tumour tissue was 55.9±2.1 in the control group and 75.4±2.7 in the rhEPO group, P<0.001. The increased microvessel diameter in rhEPO-treated animals was more pronounced in the tumour core  than in the tumour rim , Overall microvessel diameter . Within each group, the difference in VEGF expression in the tumour core vs tumour rim was not significant.Immunohistochemistry data are summarised in α did not differ significantly between both groups . There was also no significant difference in expression of Bax  or Bcl-2  between control and rhEPO-treated animals. In rhEPO-treated animals, Bcl-2 expression was significantly lower in the tumour core compared with the tumour rim .Total expression of HIF1The presence of hypoxia adversely affects radiotherapy response and prognosis in cancer patients . As the in vivo effects of RT on colorectal cancer microvasculature. Undoubtedly, modulation of RT effects is mediated by both increased oxygenation and direct effects of rhEPO on normal and tumour microvessels. Moreover, solid tumours are characterised by an important heterogeneity with both well oxygenated and hypoxic or necrotic regions. Therefore, noninvasive functional imaging was used that allows differentiating between different tumour regions.We aimed to study how rhEPO modulates the early et al found a positive correlation between tumour hypoxia and EPO-R expression, whereas others did not observe any correlation in a similar patient cohort (Expression of the EPO-R was present in all tumours and significantly more pronounced in the hypoxic tumour core. There is at present no clearly defined relationship between tumour hypoxia and expression of the EPO receptor by cancer cells. In head and neck cancer patients, Arcasoy The experiment was not intended to study changes in macroscopic tumour growth. We analysed rhEPO-mediated modulation of tumour cell sensitivity to apoptosis. In contrast to the findings of Dynamic MRI with a macromolecular CA is a validated technique to provide a comprehensive assessment of tumour microvascular physiology (In keeping with previous preclinical and clinical findings (α expression was noted, the difference in VEGF expression is likely to result both from better oxygenation of the tumour rim and from direct effects of rhEPO on VEGF expression. Radiotherapy itself has been shown to induce changes in tumour tissue oxygenation. After a single dose of 20 Gy, This modulation of RT effects was accompanied by a significantly lower expression of VEGF in rhEPO-treated animals, a finding previously reported in colorectal xenografts (Erythrocyte flux was measured with the laser Doppler shift method and, in contrast to the modelled plasma flow, provides additional information on rheological properties of the microvasculature. In rhEPO-treated animals, RT did not result in a decrease in LBF, whereas in the central tumour region of control animals, LBF was reduced by RT. This difference in LBF response probably reflects the observed difference in microvessel diameter.In keeping with others (In conclusion, the effects of RT on colorectal tumour microvascular physiology are spatially heterogeneous and modulated by administration of rhEPO. Treatment with rhEPO prevented RT-induced changes in microvascular permeability and tumour vascular volume, accompanied by a larger microvessel diameter and altered spatial complexity compared with control animals. It is at present unclear whether this microvascular modulation increases or counteracts the antitumoural efficacy of RT in this model. The present experiment does not allow to draw any conclusion on the effects of rhEPO on radiation response of the tumour cell population as opposed to the response of tumour microvessels. However, as rhEPO resulted in an increased oxygenation of the tumour rim, radiation response of the clonogens is likely to be increased by rhEPO, as described by others (Further preclinical experiments will have to elucidate the multiple molecular effects of rhEPO and the interaction with RT on tumour cells, neoplastic endothelium, and normal endothelium.


N-ethyl)phenyl)-3--6-benzylidene-1-methyl-2,5-dioxo-3-piperazinylidene)methylbenzamide) is a potent and specific inhibitor of P-gp, which reverses drug resistance in several murine and human MDR cell lines. In this study we have evaluated the in vivo efficacy of this novel modulator in a panel of murine and human tumour models and examined its pharmacokinetic profile. Efficacy studies in mice bearing MDR syngeneic tumours  or human tumour xenografts  demonstrated that co-administration of XR9051 significantly potentiated the anti-tumour activity of a range of cytotoxic drugs. This modulatory activity was observed following parenteral and oral co-administration of XR9051. In addition, the combination schedules were well-tolerated. Following intravenous administration in mice, XR9051 is rapidly distributed and accumulates in tumours and other tissues. In addition, the compound is well-absorbed after oral administration. These data suggest that XR9051 has the potential for reversing clinical MDR mediated by P-glycoprotien. © 1999 Cancer Research CampaignOverexpression of P-glycoprotein (P-gp) is a potential cause of multidrug resistance (MDR) in tumours. We have previously reported that XR9051 (


The subcutaneous growth of the Lewis lung tumour in C57BL mice chronically exposed to fresh cigarette smoke was increased above that in age-matched control mice. When murine sarcoma virus (Harvey) induced tumour cells were introduced to the lungs of groups of BALB/c mice, only mice chronically exposed to fresh cigarette smoke died with tumour cells in the lungs. Tumour cell growth in mice during short term cigarette smoke exposure was indistinguishable from that in controls.


In this study, we have examined the relative expression of clone 4-truncated, exon 5-deleted and exon 7-deleted oestrogen receptor-α variant mRNAs in 15 primary breast tumour samples and in synchronous axillary lymph node metastases. Overall, there were no significant differences between the primary tumours and the matched metastases in the relative expression of these three specific variant mRNAs. Furthermore, the pattern of all deleted oestrogen receptor-α variant mRNAs appeared conserved between any primary and its matched secondary tumour.


Tourniquet anoxia increased radioresistance of growth of primary tumour by (OER) factors of 2·9-3·3. Corresponding factors for inhibition of growth of metastases in abdominal lymph nodes, and for the reduction in incidence of lung metastases produced by single tumour cells, were 2·7 and 2·4 respectively. These results suggest that this tumour was radiobiologically well oxygenated when it was irradiated in a poorly vascularized stage of growth where tumour necrosis had developed.The effect of local irradiation of a rapidly metastasizing sarcoma in the leg of the rat was measured in terms of (a) regression of the primary tumour and (b) growth of metastases produced in lymph nodes and lungs, by dissemination occurring after irradiation of the primary tumour. These effects on rats which had been irradiated while breathing air were compared with rats breathing 10% O


The potential use of human head and neck (H & N) tumours, growing in athymic nude mice, for preclinical assessment of cytostatic drug sensitivity in a soft agar cloning system was examined. Of 20 H & N tumour xenografts, obtained from 6 different xenograft lines, 17 demonstrated sufficient colony growth to evaluate in vitro drug sensitivity. Moreover, all xenografts provided enough cells to test 8 cytostatic drugs at 3 concentrations each. A dose-dependent inhibition of colony growth was obtained with all drugs tested, except methotrexate. Tumours were considered sensitive when the drug concentration required to inhibit colony formation by 50%, was less than 1/10 of the peak plasma concentration in patients. All H & N tumour lines were resistant to cisplatin, doxorubicin, hydroxyurea, mafosfamide  and methotrexate. Bleomycin was active in 1/6 and 5-fluorouracil in 6/6 of the H & N tumour lines tested. In 32 cases the in vitro data of the H & N tumour lines and a chemosensitive rat rhabdomyosarcoma were compared directly with in vivo results obtained in nude mice. The clonogenic assay correctly predicted sensitivity in 4/6 (66.7%) and resistance in 21/26 (80.8%) of the cases. A lack of correlation was noted for methotrexate, 5-fluorouracil and cyclophosphamide. In vitro culture of human H & N xenografts may provide a means for a rapid and large scale screening to identify new drugs active against H & N malignancies. In addition the clonogenic assay may help to select drugs for subsequent testing in the nude mouse xenograft model. The lack of correlation for some drugs in the present study indicates that there are some limitations in the use of xenograft tumour material for in vitro testing of new drugs.


Therefore, chronic hypoxia may potentially be exploited to increase tumour cell radioresponse through the cytokine-inducible iNOS pathway. © 2001 Cancer Research Campaign http://www.bjcancer.comChronic hypoxia up-regulated the mRNA and protein expression of inducible nitric oxide synthase (iNOS) in EMT-6 tumour cells exposed to interferon (IFN)-gamma and interleukin (IL)-I beta. Low concentrations of cytokines (1 unit ml


DCC - and c-erbB-2 genes in disseminated breast cancer cells and actuarial relapse-free survival. Furthermore, increasing numbers of genomic imbalances measured in disseminated tumour cells were significantly associated with worse prognosis of recurrent disease. Logistic regression and Cox multivariate analysis led to the identification of genomic imbalances as an independent prognostic factor. Determination of disseminated tumour cells by genotyping of oncogenes and tumour suppressor genes seems not only to be a useful adjunct in follow up of carcinoma patients but provides also valuable additional individualized prognostic and predictive information in breast cancer patients beyond the TNM system. © 2000 Cancer Research Campaign http://www.bjcancer.comThe prognostic value of disseminated tumour cells derived from 353 breast cancer patients was evaluated. Disseminated tumour cells were purified from blood using a newly established method and nucleic acids were subsequently isolated. We investigated genomic imbalances (GI) such as mutation, amplification and loss of heterozygosity of 13 tumour suppressor genes and 2 proto-oncogenes using DNA from isolated minimal residual cancer cells. Significant correlations were found between genomic alterations of the


Clinical anti-tumour efficacy of anti-cancer drugs is a function of dose intensity, i.e. the concentration--time profile in tumour tissue. Hence, information on drug concentration profiles in tumours is of critical importance but appropriate methods for measurement are lacking. The aim of the present study was to obtain, by microdialysis sampling, concentration--time profiles in a solid tumour (melanoma) of a model anti-cancer drug, carboplatin, and thereby to assess the scope of microdialysis for tumour pharmacokinetic studies in man. Six patients with cutaneous melanoma metastases at the extremities or body trunk, scheduled to receive carboplatin (400 mg m-2 i.v.) were studied. Carboplatin concentrations were monitored in serum, intratumoral and subcutaneous tissue. Calibration of the microdialysis probes was carried out in vitro and in vivo with use of the retrodialysis method. Complete carboplatin concentration vs time profiles in tumour and subcutaneous tissue were obtained. Major pharmacokinetic parameters  were calculated for tissues and tumour/serum concentration ratios for carboplatin were derived. Mean free concentrations of carboplatin in cutaneous melanoma metastases reached only about 50-60% of total serum levels; maximal intratumoral concentrations were 7.6  microgram/ml, mean concentrations in subcutaneous tissue were similar to those in tumour. The present study demonstrates that microdialysis is a novel tool for measuring drug concentrations in solid tumours in humans in vivo and appears to be a valuable addition for pharmacokinetic/pharmacodynamic studies in oncology.


The post-natal development of “natural” resistance of Balb/c mice to the challenge of syngeneic tumours was studied using injections of various doses of live neoplastic cells into untreated animals of increasing age, from neonatal to 12 weeks.The minimum quantity of neoplastic cells capable of inducing tumours increased in parallel with the age of the animals. Immunodepression with whole body irradiation with X-rays reduced the resistance offered by adult mice to tumour challenge to that of the neonate. The relationship of the increase in resistance to tumour challenge with the development of the animals' own immune response during the course of post-natal growth is discussed.


Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) contains crucial information about tumour heterogeneity and the transport limitations that reduce drug efficacy. Mathematical modelling of drug delivery and cellular responsiveness based on underutilised DCE-MRI data has the unique potential to predict therapeutic responsiveness for individual patients.To interpret DCE-MRI data, we created a modelling framework that operates over multiple time and length scales and incorporates intracellular metabolism, nutrient and drug diffusion, trans-vascular permeability, and angiogenesis. The computational methodology was used to analyse DCE-MR images collected from eight breast cancer patients at Baystate Medical Center in Springfield, MA.Computer simulations showed that trans-vascular transport was correlated with tumour aggressiveness because increased vessel growth and permeability provided more nutrients for cell proliferation. Model simulations also indicate that vessel density minimally affects tissue growth and drug response, and nutrient availability promotes growth. Finally, the simulations indicate that increased transport heterogeneity is coupled with increased tumour growth and poor drug response.Mathematical modelling based on DCE-MRI has the potential to aid treatment decisions and improve overall cancer care. This model is the critical first step in the creation of a comprehensive and predictive computational method. The ability to predict the response of a patient to chemotherapeutic treatment is necessary to identify correct therapeutic regiments and dosages . AbnormaKtrans) has been shown to correlate with therapeutic responsiveness , which measures the accessibility of small molecules to different tumour regions  because siveness . ChangesTo obtain predictions for the therapeutic response of individual patients, one needs to interpret spatially dependant transport information from DCE-MRI. Mathematical models that include molecular transport, cell growth, and drug response have the potential to interpret DCE-MRI data and provide a basis for patient-specific predictions. Previous mathematical models have predicted tumour growth rate based on the mechanisms of nutrient diffusion and cell growth and death . Tumour μm), vascular (100 μm), voxel (mm), and whole tumour (cm); and multiple time scales, including cellular diffusion (seconds), gadolinium infusion (minutes), and tumour drug response (weeks). The model was used to analyse DCE-MR images and generate predictions for eight patients at Baystate Medical Center in Springfield, MA. Because of its mechanistic basis, we believe that this theoretical framework has potential to be a predictive therapeutic tool for individualised therapy.Here we describe a mathematical framework that incorporates DCE-MRI data into a tumour growth model to predict therapeutic response. We have used this computational method to test the hypothesis that local transport properties can predict therapeutic response. Specifically, we hypothesise that (1) increased trans-vascular transport predicts improved theoretical response, and (2) increased spatial heterogeneity predicts reduced theoretical response. The drug response model is based on our previous tumour models that integrate intracellular metabolism, nutrient and drug diffusion, cell-cycle progression, cellular drug effects, and drug pharmacokinetics . The cur−1 Gd-DTPA, and (3) scanning an additional six times, with a spacing of approximately 2 min. Ethical approval for the study was granted by the institutional review board at Baystate Health Systems (IRB07-013). All data were acquired before the study as part of standard care procedures and identifying information was removed to protect patient confidentiality (as per 45 CFR 46.101(b)). The study followed all HIPAA guidelines.Included in this study were eight female breast cancer patients who were referred to the Baystate Breast MRI Center between March 2006 and May 2007. Patients, with ages ranging from 40 to 73, were selected to include a diverse population based on tumour staging and aggressiveness. The imaging procedure consisted of (1) scanning each patient to establish an unenhanced image, (2) administering 0.1 mmol kgThe procedure for determining therapeutic response from DCE-MRI contained three major steps. First, a theoretical framework was developed and simulations were run to generate relations between transport parameters and local drug response . Second,μm rectangular cuboids whose size is specified by spectrometer resolution. Because the average spacing between blood vessels in tumours is much less than these limiting dimensions, a set of assumptions were made about the tissue inside each voxel. These assumptions were necessary to interpret DCE-MRI transport information and couple the disparate length scales. The primary assumption was that the tissue within each voxel was uniform. Although this is a considerable simplification, it is the only way to connect MRI data to therapeutic predictions without specific information about the vasculature geometry within each voxel. To implement this assumption, we assumed each voxel to contain multiple identical repeating Krogh tumour vessel cords  cell growth, which increased cord width; and (2) angiogenesis, which increased the number of cords . The ratω. Angiogenesis was controlled by the diffusion of angiogenic factors , produced by tumour cells in hypoxic microenvironments , were calculated for each voxel in a tumour as described below; (2) transport parameter pairs were converted into equivalent parameter pairs of angiogenic sensitivity, α, and average vessel permeability, P; (3) α and P parameter pairs were used to determine the local rate of cord growth, μG, and the relative decrease in volume due to drug administration, ΔV; and (4) local rates were summed over all included voxels to obtain overall tumour values.Overall tumour growth and drug response dynamics were determined by a four-step process : (1) locKtrans, fV) pair there existed one unique  pair and one unique  pair. The implementation of parameter mapping rendered tumour analysis a rapid matrix multiplication and element summation process. Parameter maps were generated by simulating tumour cords with all physiological feasible values of α and P and determining the corresponding values of Ktrans, fV, μG, and ΔV. The α and P grid consisted of 3600 points (60 on a side) spanning α values from 1 × 10−5 to 1 × 101 mM per day and P-values from 1 × 10−2 to 1 × 102 cm min−1.To streamline tumour analysis, we generated maps between parameter pairs. For each . Tumour growth rate, Ktrans and the vascular volume fraction, fV /I0 /I0  the sum I−I0)/I0 . Mass flfV and Ktrans, were tuned until calculated enhancement profiles best matched measured profiles. Parameter estimation was performed using least-squares analysis and the Matlab optimisation routine fmincon. Tumour boundaries were defined as regions with fV >2% or Ktrans >0.005 min−1. All completely surrounded interior regions were included and disconnected regions 5 pixels and smaller were excluded. Methods for the construction of parameter maps and whole tumour images are described in The signal enhancement profile of every voxel in every image was analysed individually using iterative optimisation. Values for the two adjustable parameters, Ktrans and fV. These values were subsequently converted into average growth rate and drug response.The effect of heterogeneity was investigated by comparing predicted results for whole complex tumours to those with averaged transport properties. The behaviour of hypothetical homogeneous tumours was determined using a technique similar to that currently used clinically: a tumour signal enhancement was determined by averaging the signal enhancement of all voxels within a defined tumour boundary. The resulting signal enhancement time profile was analysed as described above to obtain average values for Ktrans variance. Ktrans was assumed to have a β-distribution with a mean value of 0.015 min−1, which is the median value in the patient population. The relative variance was increased from 0.1 to 1.75 to include all values observed in the measured tumour set. The generation of hypothetical heterogeneous tumours is described in Ktrans variance in the patient population, we normalized variances to eliminate the effect of different mean values. Variances were normalised by average Ktrans values and responses were normalised by the growth rate and drug response of homogeneous tumours with the same mean.To systematically test the effect of transport heterogeneity, we calculated growth rate and drug response for a set of hypothetical tumours with increasing The existence of statistically significant correlations was determined by linearly regressing data and using one-factor ANOVA. A correlation was significant if the slope of the regression was significantly different from zero. Confidence intervals greater than 95% were considered significant.α and P , the transport parameters , and the local responses . Ultimately, direct maps between the directly measured transport parameters  and local responses  were created  the transport parameter, Ktrans, had the strongest correlation with the predictive responses  higher growth rates and better responses to chemotherapy  and decreased drug response  and because they begin to provide a mechanistic connection between imaging data and drug efficacy.Here we describe a predictive multi-scale tumour model that incorporates drug transport, angiogenesis, pharmacokinetics, and intracellular metabolism. A method was developed to rapidly determine transport parameters from individual patient DCE-MRI acquired in a clinical setting. Angiogenesis was modelled by extending the Krogh model to include formation of new blood vessels. The model framework introduced a new parameter, Ktrans strongly correlates with overall survival and Ktrans heterogeneity can distinguish between responding and non-responding patients  and Ktrans  or greater leakiness (high P). At any given Ktrans value, high vessel density was associated with low permeability, and low density was associated with high permeability  vessel density minimally affects tissue growth and drug response, (2) nutrient availability drives tissue growth, and (3) angiogenic rates vary across tumours. All the results suggest that DCE-MR images contain critical information that can predict therapeutic efficacy and that mathematically modelling has the potential to improve treatment decisions. We believe that this model is a first step towards the creation of a comprehensive computational method that will provide individual therapeutic predictions based on imaging data. Continued development of these methods has the potential to provide individualised patient predictions and improve overall cancer care.We have developed a model framework that integrates individual patient data into a tumour growth model, which accounts for intracellular metabolism, nutrient and drug diffusion, trans-vascular permeability, and angiogenesis. To the best of our knowledge this is the first report on the acquisition of transport parameters from individual patient DCE-MRI and their use in a predictive computational model. Simulations with this model suggested that