Two weeks of chronic morphine treatment at clinically relevant doses stimulates COX-2 and PGE2  and angiogenesis in breast tumours in mice. This is accompanied by increased tumour weight (∼35%) and increased metastasis and reduced survival. Co-administration of celecoxib prevents these morphine-induced effects. In addition, morphine and celecoxib together provided better analgesia than either agent alone. Celecoxib prevents morphine-induced stimulation of COX-2, PGE2, angiogenesis, tumour growth, metastasis and mortality without compromising analgesia in a murine breast cancer model. In fact, the combination provided significantly better analgesia than with morphine or celecoxib alone. Clinical trials of this combination for analgesia in chronic and severe pain in cancer are warranted.Morphine and its congener opioids are the main therapy for severe pain in cancer. However, chronic morphine treatment stimulates angiogenesis and tumour growth in mice. We examined if celecoxib (a cyclooxygenase-2 (COX-2) inhibitor) prevents morphine-induced tumour growth without compromising analgesia. The effect of chronic treatment with celecoxib (by gavage) and/or morphine (subcutaneously), or PBS on tumour prostaglandin E Mice were euthanised using compressed CO2 from the gas cylinders at the conclusion of the duration of the treatment and/or if they appeared moribund (unable to reach food and water and impaired mobility).Eight- to twelve-week-old A/J mice  were maintained under controlled environmental conditions  in our AAALAC-accredited facility and provided with standard laboratory food and water Pharmacological-grade morphine , and Celecoxib  were used. All other chemicals were obtained from Sigma Chemicals , and cell culture reagents were obtained from Invitrogen unless otherwise mentioned.3 in 12 days, and they are highly vascular, invasive and metastasise to the lungs, resulting in the death of the animals starting at about 15 days after tumour-cell injection. SCK tumour cells (2 × 105) in 0.05 ml of serum-free medium were injected subcutaneously into the right hind thigh of A/J mice. Palpable and measurable tumours appeared 4–5 days after injection.SCK mammary carcinoma was generated in rodents using breast cancer cells derived from a tumour that spontaneously arose in a female A/J mouse (Jackson Laboratories) in our laboratory  normal saline s.c. at 50 μl and 0.5% methylcellulose at 100 μl by gavage (control); (b) morphine sulphate s.c. at 0.714 mg per kg per mouse per day for 7 days and 1 mg per kg per mouse per day thereafter  and 0.5% methylcellulose at 100 μl by gavage to ensure similar administration of vehicle in each animal; (c) celecoxib at 30 mg per kg per mouse by gavage in 0.5% methylcellulose and (d) morphine sulphate plus celecoxib as described in b and c.To examine the effect of celecoxib on tumour PGEμl and 0.5% methylcellulose at 100 μl by gavage; (b) morphine sulphate s.c. at 0.714 mg per kg per mouse per day for 7 days and 1 mg per kg per mouse per day thereafter; (c) celecoxib at 100 mg per kg per mouse by gavage in 0.5% methylcellulose and (d) morphine sulphate plus celecoxib as described in IIb and IIc.To determine the effect of high-dose celecoxib, mice were treated with (a) normal saline s.c. at 50 Tumour cryosections were immunostained with the endothelial cell marker anti-CD31-PE  and the nuclear stain DAPI , as described previously  for 60 min before the test . Membranes were blocked for 1 h at room temperature in 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween 20, and incubated with 1 : 500 dilution of anti-COX-2 antibody  or anti-β-actin  at 4°C overnight. After washing, membranes were incubated with 2° species-specific antibodies conjugated to alkaline phosphatase for 45 min at room temperature. Proteins were visualised with an ECF Western blotting system . Chemiluminescent signals were acquired using a Storm 860 PhosphorImager .Whole tumour lysates containing 50 Total RNA was isolated from tumours using Trizol reagent  as per the manufacturer's instructions. Lysates were purified using acetone precipitation of proteins, and supernatants from each sample were quantified using two different dilutions of each. In this method, the intensity of the colour produced is measured at 412 nm and is inversely proportional to the amount of PGE2 present in the well. Recovery of PGE2 was calculated based on the recovery of a cold spike of PGE2 added to the lysis buffer and processed in parallel with tumour lysates. Prostaglandin E2 content was calculated using a calibration curve prepared with 10–1000 pg per ml of PGE2.Prostaglandin ELungs were fixed in 10% formalin overnight, and paraffin-embedded lung sections were stained with haematoxylin and eosin. Metastatic foci were visualised in a double-blinded manner using a light microscope.Duration of survival in the four treatment groups was analysed with Kaplan–Meier curves. Equality of survival curves was assessed using the Wilcoxon's log-rank test, which weights differences in the curves by the number of surviving mice .Tumour weight was analysed with an ANOVA model, and pairwise comparisons among the four treatment groups were made using Tukey's studentised range test (HSD) .Four measures of vascular complexity  were performed at three sites in three mice from each treatment group. Mice were randomly selected from among those living at the end of 14 days. Differences between treatment groups of these measurements were assessed using an ANOVA model with a random intercept for each mouse to account for within-mouse correlation of measurements. Tests for treatment group effects used the between-within method for determining the degrees of freedom and maximum likelihood to fit the model  in tumours of mice treated with morphine . Combined treatment of celecoxib with morphine significantly reduced all angiogenic parameters as compared to morphine by itself. Tumours in the celecoxib-treated group had lowered vessel density, number, length and branching as compared to controls, but there was no statistically significant difference. Taken together, these data suggest that morphine stimulates tumour angiogenesis in SCK tumours similar to that shown for MCF7 human tumours in nude mice . Furthermore, celecoxib-treated mice showed no significant difference in their tumour weights as compared to PBS-treated controls after 13 days of treatment with celecoxib. Interestingly, tumours in morphine and celecoxib co-administered mice were significantly smaller than PBS-treated mice (P<0.001).We next evaluated the effect of morphine and celecoxib on tumour growth in tumour-bearing mice. After 13 days of treatment with morphine, mean tumour weight in morphine-treated mice was 1.35 g compared to 0.95 g in PBS-treated controls P<0.001; . CelecoxIncreased angiogenesis and tumour growth after 13 days of morphine treatment was accompanied by increased metastasis . A signiWe examined the effect of morphine and celecoxib on survival in tumour-bearing mice. Treatments were started 1 day after tumour cell injection and continued for 14 days. The day of death represents the number of days after the initiation of treatments. Morphine-treated mice had the worse survival rate with 70% dying by day 14 as compared to 30% of PBS-treated controls . Only 20In the SCK tumour model, tumours at day 4 are barely palpable and non-metastatic. Therefore, the early death of mice after celecoxib treatment is not due to cancer progression. Several studies on rodents have used celecoxib in the range 30–250 mg per kg per mouse for several days to weeks ; both groups had palpable and measurable tumours on day 5. Morphine alone had an anti-nociceptive effect after 5 days of treatment (P<0.003 vs PBS-treated control), but had no effect after 10 and 14 days of treatment as compared to controls. In contrast to the effect of celecoxib or morphine alone, the co-administration of both resulted into a continuous analgesic effect for the entire 14 days of treatment. Paw withdrawal latencies in this group were no different than the baseline throughout the 14-day period. The duration of heat tolerance was significantly higher when both drugs were co-administered as compared to the effect of all other treatment groups (P<0.0001 on days 10 and 14 for morphine+celecoxib vs morphine or celecoxib or control). Although celecoxib treatment resulted in decreased latency vs control on day 10 (P<0.003), there was no statistically significant difference between morphine and control on day 10. On day 14, PWLs decreased further after morphine or control treatment as compared to day 10, and were no different than celecoxib treatment on day 14. Injections during the course of treatment for all groups did not result in any noticeable abnormal behaviour.Treatment effects on pain were tested using the PWL of the tumour-bearing leg, using a modified Hargreaves device. Baseline PWL was determined on day 0 before starting the treatments and 24 h after injecting the tumour cells. This time was chosen to avoid recording effects of pain due to the injection procedure and inflammation that could occur immediately after injection. Mice had no signs of swelling, inflammation or impaired mobility 24 h after tumour-cell injections. Baseline PWLs were the same for all treatment groups. Paw withdrawal latencies were significantly decreased in mice after 5 days of treatments (or 6 days after tumour-cell injection) with PBS and celecoxib , impaired analgesia and increased tumour angiogenesis, growth, metastasis and mortality. We show that inhibition of COX-2 by co-administration of celecoxib prevents morphine-induced tumour growth and metastases and increases survival. Furthermore, co-administration of celecoxib with morphine provides analgesia even after 14 days of treatment, suggesting that celecoxib may have a salutary effect on opioid analgesia. Cyclooxygenase-2 inhibition may at least partly explain the clinical improvements in postoperative pain control that have been seen with a combination of opioids and COX-2 inhibitors and/or NSAIDs -derived peptide  . SimilarThe loss of mice 13 days after morphine treatment appears to be related to increased tumour growth and metastasis as compared to PBS-treated mice. In contrast, mice treated with celecoxib plus morphine showed significantly reduced tumour growth as compared to PBS or celecoxib-treated mice. Immunofluorescent images of tumour sections show appreciably wider vessels in both groups of mice treated with morphine or morphine plus celecoxib. As morphine appears to induce tumour vessel dilation, we speculate that it may facilitate the availability of celecoxib in the ischaemic tumour. Earlier observations have shown that morphine induces vasodilatation by stimulating NO , and growth factors such as VEGF have been shown to upregulate MOR expression in endothelial cells (Some studies have demonstrated inhibition of angiogenesis by high doses of morphine and Our observations are in agreement with a study by In conclusion, pain is closely linked to disease progression and metastasis in cancer. Therefore, finding suitable strategies to combat both without having a deleterious effect on one or the other is necessary in order to provide maximum benefit to the patient, resulting in better quality of life and improved survival. Considering that opioids are the only choice of treatment for severe pain, the addition of celecoxib to cancer-related pain management regimens may improve the analgesic response to morphine and also prevent morphine-induced angiogenesis, tumour growth and metastasis. Therefore, the hypothesis that has been proved in mice in this study warrants human trial to examine its application in the management of pain in cancer patients.


An in vivo study of tissue distribution kinetics and photodynamic therapy (PDT) using 5-aminolaevulinic acid (ALA), chlorin e6 (Chl) and Photofrin (PII) was performed to evaluate the selectivity of porphyrin accumulation and tissue damage effects in a tumour model compared with normal tissue. C26 colon carcinoma of mice transplanted to the foot was used as a model for selectivity assessment. Fluorescence measurements of porphyrin accumulation in the foot bearing the tumour and in the normal foot were performed by the laser-induced fluorescence (LIF) system. A new high-intensity pulsed light delivery system (HIPLS) was used for simultaneous irradiation of both feet by light in the range of 600-800 nm, with light doses from 120 to 300 J cm-2 . Photoirradiation was carried out 1 h after injection of ALA, 3 h after injection of Chl and 24 h after injection of PII. A ratio of porphyrin accumulation in tumour vs normal tissue was used as an index of accumulation selectivity for each agent. PDT selectivity was determined from the regression analysis of normal and tumour tissue responses to PDT as a function of the applied light dose. A normal tissue damage index was defined at various values  of antitumour effect. The results of the LIF measurements revealed different patterns of fluorescence intensity in tumour and normal tissues for ALA-induced protoporphyrin IX (ALA-PpIX), Chl and PII. The results of PDT demonstrated the differences in both anti-tumour efficiency and normal tissue damage for the agents used. The selectivity of porphyrin accumulation in the tumour at the time of photoirradiation, as obtained by the LIF measurements, was in the order ALA-PpIX > Chl > PII. PDT selectivity at an equal value of anti-tumour effect was in the order Chl > ALA-PpIX > PII. Histological examination revealed certain differences in structural changes of normal skin after PDT with the agents tested. The results of PDT selectivity assessment with respect to differences in mechanisms of action for ALA, Chl and PII are discussed.


The effect of anti-enzyme antibody clearance on prodrug turnover in antibody-directed enzyme prodrug therapy (ADEPT) has been studied. Mice bearing LS174T xenografts were given localising carboxypeptidase G2 (CPG)2 conjugate (AEC) and 19 h later galactosylated anti-CPG2 antibody (SB43-GAL). In regimen I prodrug was injected 5 h after SB43-GAL as previously described. In regimen 2 and 3 a shortened and extended clearance time was used in which prodrug was administered 0.5 h or 53 h after SB43-GAL respectively. Regimen 1 resulted in similar tumour and normal tissue levels of active drug to those of the control in which prodrug was given 72 h after AEC. SB43-GAL therefore accelerated clearance of enzyme allowing early administration of prodrug. In regimen 2, very high active drug levels were found in the liver, showing removal of AEC from the blood followed by reactivation of enzyme and extensive and rapid prodrug turnover. Active drug levels in tumour and blood reached similar peak levels to those of the control. Regimen 3 resulted in lower active drug levels in tissues, consistent with degradation and excretion of enzyme. Regimen 3 also produced the best tumour to normal ratios for active drug. Residual prodrug in tumour was unaffected by SB43-GAL, showing the advantage of galactosylation in minimising inactivation of CPG2 in tumour. By contrast, residual prodrug in blood persisted for longer when SB43-GAL was used. Circulatory clearance of enzyme with SB43-GAL allows prodrug to be administered expediently with reduced toxicity and with the prospect of increasing the dosage.


P = 0.0005). There was a significant inverse correlation between the tumour VMI and nodal status  and between high VMI and low thymidine phosphorylase (TP) expression . No significant association between VMI and tumour size, oestrogen receptor, epidermal growth factor receptor, grade, angiogenesis, patient age, or E-selectin was seen. There was a significant reduction in relapse-free survival (P = 0.01) with high angiogenesis. These findings show that the VMI gives new information on the mechanism of tumour angiogenesis independently from microvessel quantitation, there is a wide variation in the differentiation of tumour vasculature but the degree of capillary differentiation is not associated with quantitative angiogenesis. The VMI identifies a subset of patients who have a high chance of regional node involvement. © 2000 Cancer Research CampaignAngiogenesis, the formation of new vessels, has been demonstrated to be an indicator of prognosis in breast cancer patients. The extent of differentiation of the tumour vessels may affect access of peripheral white cells and egress or invasion of tumour cells. This has not been assessed in relation to tumour microvessel density or other variables and may be a marker of vascular remodelling. LH39 is a monoclonal antibody recognizing an epitope located at the lamina lucida of mature small veins and capillaries but not in newly formed vessels. To study vascular differentiation in breast tumours, we examined the vascular maturation index (VMI) in 12 normal and 50 breast carcinomas and this was correlated with different clinicopathological variables including angiogenesis. Mature vessels were defined by staining with antibodies to both LH39 and to CD31, using double immunohistochemistry, whereas immature vessels stained only for CD31. VMI was defined as the % fraction of mature vessels (LH39-positive) / total number of vessels (CD31-positive). The VMI was significantly higher in normal  than in tumours  (


Nitric oxide (NO) is an important molecule in regulating tumour blood flow and stimulating tumour angiogenesis. Inhibition of NO synthase by L-NAME might induce an anti-tumour effect by limiting nutrients and oxygen to reach tumour tissue or affecting vascular growth. The anti-tumour effect of L-NAME after systemic administration was studied in a renal subcapsular CC531 adenocarcinoma model in rats. Moreover, regional administration of L-NAME, in combination with TNF and melphalan, was studied in an isolated limb perfusion (ILP) model using BN175 soft-tissue sarcomas. Systemic treatment with L-NAME inhibited growth of adenocarcinoma significantly but was accompanied by impaired renal function. In ILP, reduced tumour growth was observed when L-NAME was used alone. In combination with TNF or melphalan, L-NAME increased response rates significantly compared to perfusions without L-NAME (0–64% and 0–63% respectively). An additional anti-tumour effect was demonstrated when L-NAME was added to the synergistic combination of melphalan and TNF (responses increased from 70 to 100%). Inhibition of NO synthase reduces tumour growth both after systemic and regional (ILP) treatment. A synergistic anti-tumour effect of L-NAME is observed in combination with melphalan and/or TNF using ILP. These results indicate a possible role of L-NAME for the treatment of solid tumours in a systemic or regional setting. © 2000 Cancer Research Campaign


To evaluate whether tumour-associated lymphangiogenesis, that is the formation of new lymphatic vessels (LVs) induced by a tumour, occurs in and around conjunctival malignant melanoma (MM).Clinical files and conjunctival specimens of 20 patients with histologically diagnosed conjunctival MM were analysed. Sections were stained with LYVE-1 and podoplanin antibodies as specific lymphatic endothelial markers and Ki67 as proliferation marker. The tumour area and the area covered by LV (LVA), LV number (LVN) and LV density (LVD) were measured within the tumour and in the peritumoural area in digital images of the specimen. The LV results were correlated with the histopathological characteristics, tumour location, recurrence rate, mitomycin C therapy and presence of metastases.+ proliferating lymphatic endothelial cells were detected in the tumour and in the peritumoural tissue up to 300 μm compared with the surrounding normal conjunctiva (>300 μm distance). There was a slightly positive correlation between the tumour size and the LVN and LVA in the 50 μm zone adjacent to the tumour. We did not find any significant correlations between LVs and histopathological and clinical characteristics , possibly due to the small sample sizes. Non-limbal tumours with involvement of tarsus or fornix showed a tendency towards a higher LVD compared with limbal tumours.LVs were detected in all specimens within the tumour and peritumourally. Significantly more Ki67Conjunctival MMs display tumour-associated LV within and around the tumour. The MM seems to induce lymphangiogenesis not only in the tumour, but also in its proximity. Malignant melanomas (MMs) of the conjunctiva are associated with significant morbidity and mortality due to high rates of recurrence and metastasis.5To date, only a few features have been recognised as prognostic factors for conjunctival MM: tumour location, expansion, relapse, multifocal location, involvement of the surgical margins and tumour depth are known prognostic factors for metastatic disease.7The primary treatment of conjunctival MM is surgical: complete excision with tumour-cell free margins represents the therapy of choice but cannot be sufficiently performed in cases of diffuse growth. Topical mitomycin C as adjunct therapy has been established,Conjunctival MMs are rich in blood vessels, which play a role in systemic haematogenous metastasis. However, the main route of metastasis of conjunctival MM is lymphogenic: ultrasonic examination of the draining lymph nodes or even surgical removal of the sentinel lymph nodes has been recommended. Up to now, it was not known whether conjunctival MMs also display significant tumour-associated lymphangiogenesis, that is whether the tumour induces the formation of new lymphatic vessels.The extent of lymph node metastasis is supposed to be a major determinant for prognosis and staging of tumours,10The purpose of this study was to determine whether conjunctival MMs also display tumour-induced lymphangiogenesis, which may represent a possible new prognostic factor. We used specific lymphatic endothelial markers to analyse the presence of lymphatic vessels (LVs) in the tumour itself and in the adjacent tissue, and correlated these data with the clinical outcome and histopathological characteristics of the tumours.Clinical files and histological sections of conjunctival MMs of 20 patients who were treated at the Department of Ophthalmology of the University Erlangen-Nürnberg, Germany, between 1987 and 2005, were analysed retrospectively.The files were screened, and the documented treatment and follow-up were taken into consideration. The clinical outcome of all patients was re-evaluated at the end of 2006 and again in 2008 by interviewing the patients’ general practitioners for any new progress of the disease since the last visit, especially for systemic metastasis.+) substrate (red reaction product) or 3,3′-diaminobenzidine . Sections were counterstained with Mayer haemalaun . Positive controls were performed on corneoscleral ring specimens and negative controls with control IgG.For staining of LVs, LYVE-1 served as a specific marker for lymphatic vascular endothelium. The preparation of the histological sections of conjunctival MMs was performed as described previously.Since LYVE-1 is also expressed on tissue macrophages,+ as the chromogen substrate). Positive controls were performed as described above.For podoplanin immunostaining, polyclonal rabbit antihuman antibody against podoplanin  was used, followed by biotinylated goat antirabbit IgG for 30 min and detection by a streptavidin peroxidase complex  as a specific marker for proliferating cells. LVs with at least five endothelial cells with nuclear Ki67 positivity were considered to be Ki67-positive.The additional topical mitomycin C treatment of conjunctival MM by eye-drops is standardised in our department as two 14-day cycles with mitomycin C 0.02% eye-drops five times a day with a 14-day break. Some patients were not treated with mitomycin C eye-drops due to allergy or refusal. To analyse the potential antilymphangiogenic effect of mitomycin C therapy, tumour specimens of patients who received mitomycin C treatment and had excisions later on during their clinical course (because of new suspect lesions) were compared with the specimens obtained before mitomycin C treatment.http://rsb.info.nih.gov/ij/download.html). Morphometric LV analysis was performed for the area of the tumour, the adjacent 50 μm zone, the mid-peripheral zone (50–200 μm), the peripheral zone (200–300 μm) and the conjunctiva more than 300 μm away from the tumour border . If the tumour-adjacent area was not completely represented on the specimen, we evaluated the area as far as represented. The tumour size was measured as the area covered by the tumour in the histological section. We determined the following parameters: (1) the LV number (LVN), (2) the area covered by LVs (LVA), (3) the LV density (LVD), determined by measuring the LVN and dividing it by the tumour cross-sectional area (mm2).Histological sections of conjunctival MMs of 20 patients were taken into consideration. Sections were analysed with a light microscope , and digital colour images were taken with a 12-bit CCD camera . Analyses were performed using CellˆF  and Image J analysing program  were performed in a standardised procedure using the statistic program InStat 3 . Analyses were performed using the non-parametric test for the Ki67 analysis and the Pearson rank correlation for the correlation of tumour area to LVN and LVA.The median age of the patients in the study was 70.4 years (43–100 years). Nine women and 11 men were treated. The MM of 13 patients was based on primary acquired melanosis (PAM); in seven patients, the origin of the MM remained unclear. The primary treatment was surgical, and 10 patients had an additional topical mitomycin C treatment. The primary tumour was located in the fornix (two patients), the tarsus and the upper lid (five patients) or at the limbus/epibulbar conjunctiva (seven patients). Six patients showed a widely disseminated tumour, including some who have had primary excision outside our department, so that the primary tumour location was not known. Ten patients showed a diffuse, five a nodular and five a mixed growing type of the MM. The histopathological characteristics were: 13 tumours of mixed cell type, five tumours of spindle cell type and two tumours of epitheloid cell type.Eight of the 20 patients showed more than five relapses during the clinical course. Five patients suffered from metastasis: one patient was diagnosed for gastric metastasis 7 years after primary diagnosis, two patients for submandibular and neck spreading after 2 and 3 years, and one patient for craniopharyngeal metastasis after 1 year, and one patient had parotical metastasis after 14 years.Using LYVE-1 and podoplanin staining, we identified LVs in all included MM specimens, both within the tumour itself and in the adjacent tissue . There wTo examine whether conjunctival MMs induce formation of new LVs, Ki67 staining was performed to detect proliferating lymphatic endothelial cells in the conjunctival MMs and the adjacent conjunctival tissue. Immunostaining with Ki67 revealed significantly more proliferating lymphatic endothelial cells in the tumour and in the directly adjacent conjunctiva compared with the peripheral zones. Non-parametric tests were performed for each zone separately with the following results concerning the ratio of Ki67-positive LVs: (1) tumour versus tumour adjacent conjunctiva (50 μm zone) p = 0.063 (not significant); (2) tumour versus mid-peripheral zone (50–200 μm) p = 0.021; (3) tumour versus peripheral zone (200–300 μm) p = 0.031; (4) tumour versus distant, presumably normal conjunctiva >300 μm from the tumour border p = 0.002 . The resWe analysed whether the extent of lymphangiogenesis in and around conjunctival MM was correlated with the tumour area. Therefore, analyses of the LV parameters LVA, LVN and LVD were performed in the tumour and in the tumour environment . Intratumoural LVN, LVA and LVD were not positively correlated to the tumour cross-sectional area  who underwent topical mitomycin C treatment after tumour excision and had subsequent excisions for tumour recurrence were analysed for LVD and LVN . In threAnalysing the location of the tumour, seven patients had limbal/epibulbar tumours, seven patients had a MM at the fornix or tarsus, and six patients had a disseminated MM. The analyses of LV parameters  did not show statistically significant results in these small sample sizes. Because of the small number of patients, we performed only descriptive analyses as a scatter plot. This study on conjunctival MM shows for the first time that conjunctival—and not only cutaneous—MMs display tumour-associated lymphangiogenesis.In our study, we found LVs in the tumour itself as well as in the peritumoural area. These erythrocyte-free LVs are stained with two new markers specific for lymphatic vascular endothelium, which are LYVE-1 and podoplanin. Most remarkably, the degree of actively proliferating Ki67 positive lymphatic vascular endothelial cells is significantly higher within the MM and in the close vicinity of the tumour compared with normal, resting conjunctival LVs more distant from the tumour site. The ratio of Ki67 positive LVs is reduced with growing distance from the tumour border, which supports the hypothesis of active tumour-induced formation of new LVs (tumour-associated lymphangiogenesis). It has been shown recently that lymphangiogenic growth factors, which are secreted by a primary tumour, can induce lymphangiogenesis.23In our small pilot study, there was no significant correlation between tumour size  and LVN and LVA in the tumour. The analyses of the tumour environment revealed a higher LVN and LVA in the 50 μm zone directly adjacent to the tumour, possibly being an indicator for active lymphangiogenesis in the tumour surroundings. We found a slightly inverse correlation between LVD in the tumour and the tumour cross-sectional area. On the one hand, this finding might be related to the two-dimensional calculations of the tumour area based on cross-sectional specimens of the tumour. On the other hand, small tumours might show a higher LVD as a signal for their starting potency of dissemination, possibly related to high secretion rates of lymphangiogenic growth factors. Another possible aspect is a centrally developing necrosis in larger tumours which might reduce the rate of lymphangiogenesis and cause reduced LVD. Furthermore, some studies describe a high interstitial pressure within tumours that promotes LV collapse;We analysed the putative impact of lymphangiogenesis on recurrence rate and metastasis but did not find any significant correlation, possibly related to the small sample sizes. Larger (prospective) studies now will have to evaluate tumour-associated lymphangiogenesis as a putative risk factor for tumour metastasis.Tumour location is one of the clinically most important prognostic predictors of conjunctival MM: non-limbal tumours show a higher incidence of initial systemic metastasis and reduced survival rates.Topical mitomycin C treatment might provide an antilymphangiogenic effect, as has been suggested in several other studies on the treatment of conjunctival MM.The prognostic importance of intra- and peritumoural lymphangiogenesis is becoming more established by a growing number of studies on several human cancers.19This study on tumour-associated lymphangiogenesis may pave the road to new targets for innovative therapeutic approaches and serve as a novel prognostic parameter in conjunctival MM. Much effort in antilymphangiogenic and antiangiogenic research has been made to develop new therapeutic approaches to inhibit tumour spreading. Recently, different assays of selective inhibition of LV growth on the ocular surface have been shown.MMs of the conjunctiva display LVs within and around the tumour. There is evidence for tumour-induced lymphangiogenesis, that is formation of newly formed LVs. These LVs may act as conduits for tumour metastasis. Lymphangiogenesis parameters such as LVD and LVN may become useful novel prognostic indicators for conjunctival MM. Novel antilymphangiogenic therapeutic strategies may help to optimise the therapy of conjunctival MM in the future.


Seven mice bearing SCC VII tumours were imaged using T1-weighted conventional spin echo magnetic resonance images before and 5 min, 2 h and 24 h after intravenous injection of 0.1 mmol/kg of HOP-9P. For the acquired images, signal intensities of the tumour, muscle and oil-phantom were measured. Then, tumor/oil and tumor/muscle signal intensity ratios were calculated. Nineteen mice were sacrificed before or after the administration of HOP-9P , and the biodistribution of manganese in the tumour, muscle, liver, blood and kidneys was measured using optical emission spectrometers and was expressed as micrograms of manganese per gram of tissue. The tumour/muscle signal intensity ratio at 24 h (3.18 ± 0.34) was significantly higher than precontrast ratio (1.77 ± 0.20) (P < 0.05). The biodistribution assessment of manganese demonstrated that HOP-9P gradually and consistently accumulated in the tumour to reach the highest concentration at 24 h (3.49 ± 1.22 μ gMn/g). It is concluded that HOP-9P is a potential tumour-specific MR contrast agent. © 2001 Cancer Research Campaign


Similar vascular effects were obtained in MAC 15 orthotopic tumours and SW620 human colon tumour xenografts treated with the prodrug. More importantly, in the orthotopic models, necrosis was seen in vascularized metastatic deposits but not in avascular secondary deposits. The possible mechanism giving rise to these effects was examined in HUVEC cells. Here cellular networks formed in type I calf-skin collagen layers and these networks were completely disrupted when incubated with a non-cytotoxic concentration of combretastatin A-4 or its prodrug. This effect started at 4 h and was complete by 24 h. The same non-cytotoxic concentrations resulted in disorganization of F-actin and β-tubulin at 1 h after treatment. In conclusion, combretastatin A-4 and its prodrug caused extensive necrosis in MAC 15A s.c. and orthotopic colon cancer and metastases, resulting in anti-tumour effects. Necrosis was not seen in avascular tumour nodules, suggesting a vascular mechanism of action. © 1999 Cancer Research CampaignThe anti-tumour effects and mechanism of action of combretastatin A-4 and its prodrug, combretastatin A-4 disodium phosphate, were examined in subcutaneous and orthotopically transplanted experimental colon tumour models. Additionally, the ability of these compounds to directly interfere with endothelial cell behaviour was also examined in HUVEC cultures. Combretastatin A-4 (150 mg kg


We have shown previously that isolated limb perfusion (ILP) in sarcoma-bearing rats results in high response rates when melphalan is used in combination with tumour necrosis factor alpha (TNF-α). This is in line with observations in patients. Here we show that ILP with doxorubicin in combination with TNF-α has comparable effects in two different rat sarcoma tumour models. The addition of TNF-α exhibits a synergistic anti-tumour effect, resulting in regression of the tumour in 54% and 100% of the cases for the BN175-fibrosarcoma and the ROS-1 osteosarcoma respectively. The combination is shown to be mandatory for optimal tumour response. The effect of high dose TNF-α on the activity of cytotoxic agents in ILP is still unclear. We investigated possible modes by which TNF-α could modulate the activity of doxorubicin. In both tumour models increased accumulation of doxorubicin in tumour tissue was found: 3.1-fold in the BN175 and 1.8-fold in the ROS-1 sarcoma after ILP with doxorubicin combined with TNF-α in comparison with an ILP with doxorubicin alone. This increase in local drug concentration may explain the synergistic anti-tumour responses after ILP with the combination. In vitro TNF-α fails to augment drug uptake in tumour cells or to increase cytotoxicity of the drug. These findings make it unlikely that TNF-α directly modulates the activity of doxorubicin in vivo. As TNF-α by itself has no or only minimal effect on tumour growth, an increase in local concentrations of chemotherapeutic drugs might well be the main mechanism for the synergistic anti-tumour effects. © 2000 Cancer Research Campaign


Oxygen is an essential regulator of cellular metabolism, survival, and proliferation. Cellular responses to oxygen levels are monitored, in part, by the transcriptional activity of the hypoxia inducible factors (HIFs). Under hypoxia, HIFs regulate a variety of pro-angiogenic and pro-glycolysis pathways. In solid cancers, regions of hypoxia are commonly present throughout the tissue because of the chaotic vascular architecture and regions of necrosis. In these regions, the hypoxic state fluctuates in a spatial and temporal manner. Transient hypoxic cycling causes an increase in the activity of the HIF proteins above what is typical for non-pathologic tissue. The extent of hypoxia strongly correlates to poor patient survival, therapeutic resistance and an aggressive tumour phenotype, but the full contribution of hypoxia and the HIFs to tumour biology is an area of active investigation. Recent reports link resistance to conventional therapies and the metastatic potential to a stem-like tumour population, termed cancer stem cells (CSCs). We and others have shown that within brain tumours CSCs reside in two niches, a perivascular location and the surrounding necrotic tissue. Restricted oxygen conditions increase the CSC fraction and promote acquisition of a stem-like state. Cancer stem cells are critically dependant on the HIFs for survival, self-renewal, and tumour growth. These observations and those from normal stem cell biology provide a new mechanistic explanation for the contribution of hypoxia to malignancy. Further, the presence of hypoxia in tumours may present challenges for therapy because of the promotion of CSC phenotypes even upon successful killing of CSCs. The current experimental evidence suggests that CSCs are plastic cell states governed by microenvironmental conditions, such as hypoxia, that may be critical for the development of new therapies targeted to disrupt the microenvironment. Oxygen concentration is tightly regulated at the tissue and cellular levels because of its essential role in many biological processes. Hyperoxia can induce the formation of reactive oxygen species (ROS), which may cause genotoxic effects or cell death. In contrast, hypoxia can have extensive downstream transcriptional effects, such as activation of pro-apoptotic and pro-angiogenic pathways. Nonpathological cells have regulatory mechanisms to quickly respond to variations in oxygen tension by the hypoxia inducible factors (HIFs). Neoplastic cells employ oxygen-sensitive activity of the HIF proteins. The HIFs also have higher basal activity in the neoplastic compartment. However, this enhanced level of transcriptional activity is not fully understood. It can be partly attributed to the state of oxygenation within a tumour, which is not consistent and behaves in a cyclical nature. The flux in oxygen tension experienced by cancer cells is an effect of several tumour characteristics such as chaotic vasculature, poor oxygen diffusion across an ever-expanding tumour, and irregular blood flow. Several studies have shown that the constant acute cycling of hypoxic regions in a tumour leads to enhanced metastatic potential and HIF transcriptional activity beyond what is typical of healthy tissue . Large regions of the tumour become hypoxic because of this spatial limitation. The immediate cellular response (mediated by HIF proteins) is the influx of new vessels to provide appropriate oxygenation to the tumour tissue. However, vessel ingrowth cannot maintain proper tissue oxygenation when faced with the rapid cellular expansion of a tumour. This constant imbalance between cell and vessel growth causes a fluctuating oxygen state. These transient states of hypoxia are a common characteristic of solid tumours. Although normal oxygen tension in healthy tissue is approximately 7% oxygen (53 mmHg), this tension in a tumour can range from physiological (∼7%) to severe (<1%) oxygen. Severe hypoxia is less frequent but commonly found surrounding areas of necrosis, which is another common characteristic of solid tumours. The immediate molecular response to low oxygen is stabilisation of the HIF proteins. Hypoxia inducible factors stimulate the expansion and migration of endothelial cells into the tumour space, which allow new vessel growth from the existing vasculature structure surrounding the tumour. The formation of these vessels supplies the rapidly expanding tumour with nutrients and oxygen. However, the vasculature is very chaotic and does not transfer nutrients and oxygen efficiently , and HIF3α. The HIFβ isoforms, also known as aryl hydrocarbon receptor nuclear translocator (ARNT and ARNT2), are constitutively and ubiquitously expressed across many cell types . HIF1α and HIF2α also bind homologous target DNA-binding sequences . In RCC, there is a biallelic inactivation of the E3 ubiquitin ligase responsible for targeting the HIFα subunits for degradation. Renal cell carcinoma patient specimens have higher activity of HIF regulated pathways such as increased angiogenesis, altered glucose uptake and metabolism, and loss of growth control by mitogenic signals. HIF1α and HIF2α have unequal roles in RCC and HIF2α is more important for disease progression. Inhibition of HIF2α suppresses in vivo tumour growth  to more physiologically relevant tension for tumours (2–5% oxygen)  pathways. The different roles and expression patterns of HIF1α and HIF2α demonstrate that their specificity is important for tumour propagation and survival. The HIFs have been demonstrated to be expressed in the cancer stem cell population, possibly promoting the stem-like phenotype and driving tumour growth.Certain disease states allow for the stabilisation of the HIFAlthough originally described in endothelial cells as regulators of migration and erythropoiesis, the HIF proteins have since been implicated in many cell processes. Specifically in cancer, they are critical for the ingrowth of vasculature to feed the rapidly expanding neoplastic compartment. As more is understood about the methods of tumour propagation, hypoxia has become a critical microenvironmental influence. Lending support to this theory is the recent evidence linking HIF expression in cancer stem cells to cell proliferation and tumour survival.α and demonstrated that the majority of tumour cells responded to hypoxia by stabilizing the HIF1α protein. In many cases HIF2α expression was not found or was no different between hypoxia and normoxia. The conclusion was drawn that HIF1α, not HIF2α, was responsible for the majority of the hypoxia response in cancer. However, recent experimental evidence from our laboratory has demonstrated differential protein and mRNA expression of HIF1α and HIF2α between the non-stem and cancer stem cell  decreased in these cells. A study from Pahlman and colleagues . Recently, it has been demonstrated that hypoxia is responsible for altering cellular phenotype by increasing proliferation, self-renewal, and the upregulation of stem genes in both stem and non-stem cancer cell populations (α component of the hypoxic response can increase the expression of stem cell-associated genes and confer tumourigenic potential to non-stem cells (α transcriptionally regulates Oct4 (α outside of the canonical hypoxic responses. By regulating pathways associated with normal stem cell pluripotency, HIF2α may alter basic genetic activity of cancer cells to a more stem-like phenotype depending on the constantly fluctuating state of oxygen in the tumour. These findings are significant in showing that not only is HIF2α a critical part of the hypoxic response in cancer stem cells, but that it can promote a more stem-like phenotype in stem and non-stem cancer cells.ulations . Specifiα promoting a more stem-like phenotype in cancer cells. In order to better understand tumour biology, new approaches to how cancer cells form the tumour are needed.Tumour cell heterogeneity is currently modelled in two paradigms: stochastic and hierarchical models. The stochastic model posits that most cells within a tumour possess the ability to self-renew to maintain phenotypic copies of themselves. In addition, many phenotypically distinct cell populations in the tumour are able to recapitulate the primary tumour in a secondary location. The hierarchical model suggests that cells within a tumour are organised in a hierarchy, similar to normal stem cells. At the top of the hierarchy are cancer stem cells, which are the only cells that are able to self-renew and propagate the tumour. The other cells in the hierarchy are phenotypically distinct from the cancer stem cell and do not possess stem-like characteristics. However, these present models do not completely fit what is known about cancer stem cell biology, especially with the recent demonstration of HIF2The cells that comprise a tumour can instead be modelled as accumulation of mutations or alterations in epigenetic regulation . In the


A 17-year-old patient with a small paratesticular embryonic sarcoma presented with symptoms of renal failure, polyuria and widespread bone metastases. Investigation revealed hypercalcaemia and uraemia without any evidence of hyperparathyroidism. The hypercalcaemia responded over a period of weeks to administration of mithramycin with initial improvement in the symptoms and metabolic derangements. Control was lost with the necrosis of intra-abdominal tumour deposits and haemorrhagic polypoid deposits in the alimentary tract. The value and hazards of mithramycin are well demonstrated by these rare complications of this type of tumour.


A high proportion of females of the C3H strain of mice develop tumours of the mammary gland which are caused by mouse mammary tumour virus (MMTV) transmitted through the milk. We have examined whether administration of mouse interferon (IFN) to nursing mothers and/or their suckling offspring only during the period of nursing, can affect the incidence of tumours developing in these animals. In two separate experiments, animals receiving IFN by direct injection while suckling, and remaining virgin showed a marked and statistically significant decrease in tumour incidence. Mice receiving the same or a tenfold higher dose of IFN while lactating showed no such reduction in tumour incidence, even if they had also received IFN while suckling. The results suggest that IFN can affect the initial establishment of the MMTV infection in suckling mice sufficiently to delay tumour development provided the animals are not exposed to the hormonal stimulus of pregnancy and lactation.


Studies of several tumour types have shown that expression profiling of cellular protein extracted from surgical tissue specimens by direct mass spectrometry analysis can accurately discriminate tumour from normal tissue and in some cases can sub-classify disease. We have evaluated the potential value of this approach to classify various clinico-pathological features in colorectal cancer by employing matrix-assisted laser desorption ionisation time of-flight-mass spectrometry (MALDI-TOF MS).k-nearest neighbours algorithm was used to build classification models either using separate training and test datasets or else by using an iterative, 'leave-one-out' cross-validation method.Protein extracts from 31 tumour and 33 normal mucosa specimens were purified, subjected to MALDI-Tof MS and then analysed using the 'GenePattern' suite of computational tools . Comparative Gene Marker Selection with either a t-test or a signal-to-noise ratio (SNR) test statistic was used to identify and rank differentially expressed marker peaks. The verses adjacent normal mucosa tissue . Unsupervised hierarchical cluster analysis classified most tumour and normal mucosa into distinct cluster groups. Supervised prediction correctly classified the tumour/normal mucosa status of specimens in an independent test spectra dataset with 100% sensitivity and specificity . Supervised prediction using 'leave-one-out' cross validation algorithms for tumour spectra correctly classified 10/13 poorly differentiated and 16/18 well/moderately differentiated tumours ; disease recurrence was correctly predicted in 5/6 cases and disease-free survival  was correctly predicted in 22/23 cases . A similar analysis of normal mucosa spectra correctly predicted 11/14 patients with, and 15/19 patients without lymph node involvement .73 protein peaks in the mass range 1800-16000Da were differentially expressed in tumour Protein expression profiling of surgically resected CRC tissue extracts by MALDI-TOF MS has potential value in studies aimed at improved molecular classification of this disease. Further studies, with longer follow-up times and larger patient cohorts, that would permit independent validation of supervised classification models, would be required to confirm the predictive value of tumour spectra for disease recurrence/patient survival. Colorectal cancer (CRC) is the second commonest malignancy and has a five-year survival rate of approximately 50% ,2. The mKRAS, BRAF and PIK3CA genes have all been shown to be of some prognostic or predictive value  [verses normal mucosa tissue (reviewed in [Markers based on circulating carcinoembryonic antigen (CEA) levels and various tumour-associated gene mutations including microsatellite instability (MSI), loss of heterozygosity of 18q, deleted in colorectal cancer (DCC), mutations in iewed in ). In parr (EGFR) ,12. In ar (EGFR)  and micrr (EGFR)  have reviewed in ). An expiewed in .As an alternative to 2D-DIGE, studies of other tumour types have also employed the technique of direct protein expression profiling of tumour/normal tissue by surface enhanced laser desorption ionisation time-of-flight mass spectrometry (SELDI-TOF) or by matrix-assisted laser desorption ionisation time of-flight-mass spectrometry (MALDI-TOF) mass spectrometry ,17. ThisIn the present study, we have evaluated the potential value of protein expression profiling of CRC tissue by MALDI-TOF mass spectrometry. In addition to comparing tumour with adjacent normal mucosa, we have investigated whether spectral profiles of tumour tissue can be used to classify various clinico-pathological features of disease. Since previous 2D-DIGE studies have reported abnormalities of protein expression profiles in tumour-adjacent normal tissue , we haveTissue samples were collected from a total of 36 patients with confirmed CRC at the time of surgical resection at Colchester General Hospital, Essex UK. All specimens were obtained following informed consent in accordance with local UK NHS Ethics Committee approval (protocol reference: MH 528). Surgically excised specimens were washed extensively in ice-cold 150 mM NaCl and samples of normal colonic mucosa (>10 cm from tumour margin) and tumour tissues were excised using a scalpel and then snap frozen and transferred to a - 80°C freezer. The total time from surgical resection to snap freezing of specimens was <30 mins.Frozen tissue samples (approximately 250 mg) were ground using a mortar and pestle and then lysed for 30 mins at 4°C in 1.0 ml of 10 mM Tris-HCl pH 7.5, 200 mM NaCl containing Protease inhibitor cocktail  and 1% N-octyl-β-D-glucopyranoside (Sigma Aldrich). The cell lysate was then centrifuged at 12,000 × g for 30 mins and the supernatant representing the solubilised fraction was removed. Protein was further purified by reversed phase hydrophobic interaction chromatography using a commercially available super-paramagnetic microparticle kit . Briefly, 10 μl of 30-35 mg/ml protein solution was adsorbed to 10 μl of beads after addition of 20 μl kit binding buffer. After three washes with 200 μl 0.1% trifluoroacetic acid, protein was eluted in 20 μl of 50% (v/v) acetonitrile (Fisher Scientific) Eluted protein was stored at 4°C for no more than 1 hr prior to matrix co-crystallisation.To facilitate reproducible co-crystallisation of protein with matrix solution, a modification of the slow crystallisation method  was usedCalibrated spectra were exported as ASCII files and were digitally processed by smoothing, de-noising, baseline subtraction and normalisation  using the 'SpecAlign' suite of spectral computational tools ,25. ValiP and false discovery rate (FDR) values [k-nearest neighbours (kNN) algorithm [vs normal using separate training and test datasets. For this purpose, two thirds of the spectra, comprised of a representative proportion of tumour and normal spectra, were randomly assigned to a training dataset, with the remaining third being used as an independent test dataset. Spectra were randomly assigned using the GenePattern 'SplitDatasetTrainTest' module [kNN algorithm was used in an iterative, 'leave-one-out' cross-validation mode. Other statistical analysis used the SPSS software.Subsequent spectral analysis was implemented in the 'GenePattern' suite of software tools  . Hierarc) values -30. The lgorithm  was used' module . Alternaverses normal protein expression profiles.Table verses normal tissue, the Comparative Gene Marker Selection algorithm [P values (Feature P) that were binned in increments of 0.05. Above P = 0.05, the representation of protein peaks was fairly evenly distributed. However, nearly 100 peaks gave a P value < 0.05, indicating that a sizable fraction of proteins detected by MALDI-TOF mass spectrometry discriminate between tumour and normal colonic tissue. Applying a threshold of P ≤ 0.01, FDR ≤ 0.05, the expression profile of a total of 73 protein peaks was significantly different between tumour and normal tissue with 57 being up-regulated in normal tissue and 16 being up-regulated in tumour tissue. Figure To quantitatively evaluate the differences between the protein expression profiles of tumour lgorithm  was applkNN algorithm [To rigorously demonstrate that tumour and normal mucosa tissue could be distinguished using their protein spectral profiles, the 64 spectra were randomly split into separate training and test datasets. The training spectra dataset was used to optimise a lgorithm  for predkNN algorithm was used to optimise a series of classification models. Since the limited numbers of datasets precluded analysis by using independent train and test spectra, the kNN algorithm was used in an iterative, 'leave-one-out' cross-validation mode. Table P = < 0.001). Additional file kNN algorithm results and Figure kNN model for disease recurrence also gave a low ROC error (0.105 - see Table P = < 0.001). Figure To determine whether the protein expression profiles of tumour tissue could be used to predict individual clinico-pathological characteristics of patients Table , the kNNkNN algorithm correctly predicted 11/14 patients with, and 15/19 patients without lymph node involvement (P = 0.001). Figure In a similar analysis of normal mucosa spectra Table , only thverses normal mucosa [kNN training model, the classification of tumour and normal tissue was correctly predicted with 100% sensitivity and specificity  in an independent test dataset. This performance compares favourably with other studies, for example in head and neck squamous cell carcinoma, in which supervised prediction using SELDI-TOF spectral data correctly classified healthy mucosa and tumour tissue with an accuracy of 94.5% and 92.9% respectively [Although previous studies employing 2D-DIGE analysis of CRC tissues have documented a number of proteins that are either up- or down-regulated in tumour l mucosa , the extl mucosa -20, MALDkNN predictive models for differentiation (0.171) and disease recurrence (0.105). Since histological differentiation stage is a characteristic that is intrinsic to the tumour tissue , the ability of the spectra to discriminate well/moderately differentiated from poorly differentiated histologies is perhaps unsurprising. The good performance of the predictive model for disease recurrence is consistent with data from several microarray expression profiling studies that have clearly demonstrated associations between patterns of tumour-associated gene expression and prognosis/treatment response [In further evaluating the potential value of spectra generated from tumour tissue for classifying various clinic-pathological characteristics of disease, we observed low ROC errors with the response ,13,14. HkNN model of normal mucosa spectra for lymph node involvement did give a low ROC error (0.212); the corresponding kNN model for tumour spectra did not show predictive value (0.391). One plausible scenario to explain the predictive value of normal mucosa spectra for lymph node involvement is that paracrine/inflammatory mechanisms, involving proximal affected lymph nodes, may induce changes to the microenvironment of tumour-adjacent mucosa.Several lines of evidence indicate that the normal mucosa from surgically resected CRC tumour specimens display abnormalities in gene and protein expression. These abnormalities have been attributed to precancerous 'field effect' changes in tumour-adjacent mucosa and have been reported to affect protein expression , CpG islAs an essential pre-requisite for marker validation, it would be highly desirable in future studies to determine the identities of candidate marker peaks in tumour tissue that discriminate different histological differentiation stages and predict disease recurrence. Our findings also indicate that similar studies using the alternative approach of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in CRC are warranted.In summary, our study has shown that direct protein expression profiling of surgically resected CRC tissue by MALDI-TOF mass spectrometry has potential value in studies aimed at improved molecular classification of this disease. Further studies, with longer follow-up times and larger patient cohorts, that would permit independent validation of predictive models, would be required to confirm the predictive value of tumour spectra for disease recurrence/patient survival.The authors declare that they have no competing interests.CCLL collected specimens, processed all samples and collated and analysed data. NW collected specimens and collated data. SM and TA contributed to the study design and in arrangements for specimen collection. JDN contributed to the study design, mass spectrometry and data analysis and wrote the manuscript. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/410/prepubExamples of raw MALDI-TOF spectral profiles. Illustrative examples shown for 2012NM and 020TClick here for fileSummary of marker peaks discriminating tumour from normal mucosa. Compilation of m/z values, ranking and statistics for 73 marker peaks.Click here for filePerformance of predictive model for discriminating tumour and normal mucosa. Summary of results of optimised k-NN algorithm on an independent test dataset.Click here for filePerformance of model for predicting poor differentiation based on tumour spectra. Summary of results of 'leave-one-out' cross-validation k-NN algorithm.Click here for filePerformance of model for predicting disease recurrence based on tumour spectra. Summary of results of 'leave-one-out' cross-validation k-NN algorithm.Click here for filePerformance of model for predicting lymph node involvement based on mucosa spectra. Summary of results of 'leave-one-out' cross-validation k-NN algorithm.Click here for file


S.c. injection of tumour cells or small pieces of tumour irradiated to a dose of 22,000 rad evoked resistance to live challenge with the same tumour (a CBA strain fibrosarcoma induced with methylcholanthrene) 14 days later. This resistance was, however, over-ridden if the challenging inoculum was sufficiently large, and did not develop if the cells were irradiated to 100,000 rad. The resistance evoked by injection of 10(6) irradiated tumour cells was impaired by i.p. injection of 1-4 mg C. parvum 5 days before, and virtually abolished by a similar injection 11 days after, the irradiated cells. The effect of s.c. injection of a mixture of 10(6) irradiated cells and C. parvum 14 days before live challenge depended on the dose of C. parvum. With 0-7 mg the development of resistance was largely but not completely abrogated; 0-35 mg resulted in a lesser degree of abrogation, and 0-09 mg or 0-02 mg had little or no effect.


Tumour hypoxia has been shown to increase mutation rate, angiogenesis, and metastatic potential, and decrease response to conventional therapeutics. Improved tumour oxygenation should translate into increased treatment response. Exogenous recombinant erythropoietin (rEpo) has been recently shown to increase tumour oxygenation in a mammary carcinoma model. The mechanism of this action is not yet understood completely. The presence of Epo and its receptor (EpoR) have been demonstrated on several normal and neoplastic tissues, including blood vessels and various solid tumours. In addition, rEpo has been shown in two recent prospective, randomized clinical trials to negatively impact treatment outcome. In this study, we attempt to characterize the direct effects of rEpo on tumour growth and angiogenesis in two separate rodent carcinomas. The effect of rEpo on R3230 rat mammary adenocarcinomas, CT-26 mouse colon carcinomas, HCT-116 human colon carcinomas, and FaDu human head and neck tumours, all of which express EpoR, was examined. There were no differences in tumour growth or proliferation (measured by Ki-67) between placebo-treated and rEpo-treated tumours. In the mammary window chamber, vascular length density (VLD) measurements in serial images of both placebo-treated and Epo-treated rats revealed no difference in angiogenesis between the Epo-treated tumours and placebo-treated tumours at any time point. These experiments are important because they suggest that the recent clinical detriment seen with the use of Epo is not due to its tumour growth effects or angiogenesis. These studies also suggest that further preclinical studies need to examine rEpo's direct tumour effects in efforts to improve the therapeutic benefits of Epo in solid tumour patients. Female Fischer 344 rats  were used for the R3230 flank tumour studies and the mammary window chamber experiments. Tumour cells were injected into the subcutis of the right quadriceps muscle for flank tumours. Following injection of tumour cells or mammary window surgery, the animals were allowed free access to food and water R3230 (rat mammary adenocarcinoma) and GFP-R3230  solution in 0.9% NaCl via an injection into the subcutis of the nape of the neck at a dose of 2000 units kg−1 thrice per week. This dosing is similar to a rEpo dose of 60 000–100 000 IU dose−1 in humans, as referenced in previous work  as a 100 unit mlIn rats, tumours were measured transversely and the volume calculated using the formula: μm serial sections of frozen tissue. Sections were fixed in 4°C cold acetone for 10 min. After endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 15 min, tumour sections were blocked with 10% donkey serum for 15 min. Sections were incubated in primary antibodies (anti-EpoR (M-20), 1 : 200 ; anti-Ki-67, 1 : 1000 ) overnight at 4°C. The location of the antibody reactions was visualized with 3, 3′-diaminobenzidine tetrahydrochloride (Sigma). For Ki-67 staining, four representative fields at × 40 objective taken from one tumour in each of six animals in both the control and Epo-treatment group were measured. Proliferation index was calculated by manually counting the ratio of Ki-67-positive nuclei to total number of nuclei in each field, and the average proliferation index for each tumour was calculated.Standard immunohistochemical techniques were used to stain for both EpoR and Ki-67. Briefly, IHC was performed using 10 in vivo angiogenesis. The epidermis around either the right third or fourth nipple was removed, leaving an intact nipple and surrounding subcutaneous tissue. The nipple was then removed at its base, exposing the nipple sinus. A 1 mm3 piece of Gelfoam  soaked in 3 μl of an R3230-GFP cell suspension of concentration 2 × 106 cells ml−1  was inserted into the nipple sinus. This was then covered with an acrylic window, which was sutured in place.The method used is the same as that described by The mammary windows were imaged serially using a Carl Zeiss MPS intravital microscope  every other day, starting on postoperative day 2 and ending on postoperative day 14. The microscope is equipped with a color video camera (Carl Zeiss ZVS-3C75DE) connected to a PC computer with a frame grabber and Scion Image software . Fluorescence epi-illumination was provided with a 100 W mercury-arc lamp  and a FITC filter (excitation 450–490 nm and emission 520 nm). Images were obtained at low magnification (objective × 5) and analyzed using Scion Image software. Vascular length density (VLD) was calculated for each image by an observer unaware of the treatment group.t-test for independent means. In all analyses, two-sided P-values less than 0.05 was considered significant.The data on hematocrit, tumour volumes, and VLD were analyzed and treatment groups compared using To establish relevance for this study, we determined the EpoR status of each of the four tumour cell lines used. All four tumour cell lines used in this experiment expressed Epo receptor by Western blot . This wavs 46.7±2.1% in the control group after 14 days (P<0.05). In the mammary window chamber experiments, EPO significantly increased the baseline hematocrit (45.8±2.6) of treated rats by 36% after 8 days and 40% at 14 days compared to placebo-treated rats .In the tumour growth studies, baseline hematocrit of mice was 46.5±1.5%. Treatment with Epo produced a hematocrit of 60.9±3.1 Tumour growth was observed in all rats transplanted with R3230 tumour cells and mice transplanted with CT-26, HCT-116, and FaDu tumour cells. Tumour volumes were similar between the EPO-treated and control groups in each of the four tumour cell lines throughout the length of the study . As measP>0.05 at all timepoints). All 20 rats tolerated the surgery well and were healthy immediately postoperatively. At postoperative day 14, 14 of 20 rats retained their mammary windows and had tumours that could be visualized. This represents a success rate of 70%, consistent with that of Shan (n=2), (2) lack of tumour growth (n=3), or (3) infection/inflammation that prevented tumour visualization (n=1).There was no statistical difference in VLD between the EPO-treated and control groups  at any tin vitro, has implicated rEpo as promoting tumour growth , intra-tumoural production of Epo might also influence tumour behavior in an autocrine/paracrine fashion. Evidence for a functional Epo-EpoR system has been reported by several groups: intratumoural and intraperitoneal injections of solube EpoR or neutralizing monoclonal antibody to EpoR resulted in tumour growth delay and inhibition of angiogenesis (in vivo tumour model. Our laboratory has extensively utilized the rodent dorsal skin flap window chamber model in determining real-time, sequential angiogenic effects of various agents (in vivo model, rEpo did not demonstrate any proangiogenic activity.There have been no reports to date precisely analyzing the angiogenic potential of therapeutic rEpo in an in vitro evidence suggests that rEpo may induce a cytoprotective phenotype in certain tumour types, which could translate into resistance to certain chemotherapeutics (in vivo insight into this seemingly confusing clinical situation. In addition, our study suggests that recent clinical results using rEpo in nonanemic patients may not be due to the direct effects of the drug on the tumour, as we did not detect any tumour growth enhancement with rEpo. Our findings warrant further preclinical and clinical investigation of rEpo in order to further clarify the risks of its use as well as optimize its known benefits.Finally, our studies offer insight into possible clinical effects of rEpo on patients receiving the drug to treat anemia. Two recent clinical trials  have sug


R2 and ΔR2*) after injection of USPIO provided an index proportional to the blood volume of the microvasculature and macrovasculature, respectively, for each tumour. The melanoma metastases were characterised by a blood volume and vessel leakage higher than both glioma xenografts. The U87 glioblastoma xenografts displayed higher permeability and blood volume in the rim than in the core. The E34 glioma xenografts were characterised by a relatively high blood volume, accompanied by only a moderate blood–brain barrier disruption. Delineation of the tumour was best assessed on post-USPIO gradient-echo images. These findings suggest that contrast-enhanced MR imaging using USPIOs and, in particular, ΔR2 and ΔR2* quantitation, provides important additional information about tumour vasculature.To enhance the success rate of antiangiogenic therapies in the clinic, it is crucial to identify parameters for tumour angiogenesis that can predict response to these therapies. In brain tumours, one such parameter is vascular leakage, which is a response to tumour-derived vascular endothelial growth factor-A and can be measured by Gadolinium-DTPA (Gd-DTPA)-enhanced magnetic resonance imaging (MRI). However, as vascular permeability and angiogenesis are not strictly coupled, tumour blood volume may be another potentially important parameter. In this study, contrast-enhanced MR imaging was performed in three orthotopic mouse models for human brain tumours (angiogenic melanoma metastases and E34 and U87 human glioma xenografts) using both Gd-DTPA to detect vascular leakage and ultrasmall iron oxide particles (USPIO) to measure blood volume. Pixel-by-pixel maps of the enhancement in the transverse relaxation rates (Δ Growth and metastasis of solid tumours are considered to strictly depend on angiogenesis . HoweverR2 and ΔR2* after injection of USPIO provides an index proportional to the blood volume of the microvasculature and macrovasculature, respectively -enhanced MRI has been demonstrated to be useful for the characterisation of the tumour vasculature  receptor 2 and the epidermal growth factor receptor, resulted in conversion to a nonangiogenic phenotype, characterised by perivascular growth along pre-existent vessels . The image acquisition protocol started with three gradient-echo scout images for slice positioning. The protocol of the Gd-DTPA contrast-enhanced MRI consisted of T1-weighted multislice gradient-echo images with TR/TE=400/6 ms, voxel size=136 × 136 × 1000 μm, 16 slices, receiver bandwidth=100 kHz, scan time=1 min 16 s. The images were acquired prior to and 1, 2, 10 and 20 min after administration of Gd-DTPA  at a dose of 0.2 mmol kg−1.Magnetic resonance imaging experiments were performed on a 7 T/200 mm horizontal-bore MR magnet interfaced to an SMIS console and equipped with a gradient insert  and spin-echo  imaging performed prior to and following administration of a USPIO blood-pool agent . Magnetic resonance imaging with USPIO was performed 2–3 h after Gd-DTPA imaging in mice with intracerebral Mel57-VEGF-A165 (n=4), E34 (n=3) or U87 (n=4) tumours. This time gap allowed complete wash out of Gd-DTPA and enabled a correlation between Gd-DTPA and USPIO enhancement.A number of pilot experiments were performed to optimise the protocol of blood volume measurements. At the dose of USPIO agent used in the current study, a good compromise between signal-to-noise ratio (SNR) and contrast enhancement was found for values of TE in the range of 5–10 ms. As susceptibility effects scale linearly with the static magnetic field strength, longer (shorter) values of TE might be advantageous for blood volume measurements at a lower (higher) field. The protocol of the USPIO contrast-enhanced MRI consisted of multislice gradient-echo  and post-USPIO (Soaft), in the gradient-echo images. The same algorithm was employed to generate ΔR2 maps from the spin-echo images. For each mouse, after ΔR2* and ΔR2 colormaps were generated, three slices were chosen, based on the maximum values of ΔR2* in tumours, as assessed on the ΔR2* colormaps. ΔR2* and ΔR2 were measured in manually segmented ROIs on these slices. For each animal carrying Mel57-VEGF-A165 tumours, at least five lesions were analysed. In the animals carrying E34 and U87 tumours, multiple ROIs (n=9 per mouse) were defined within the tumour. Similarly, ROIs were defined within healthy brain regions . For each ROI, mean ΔR2 and ΔR2* were calculated by averaging the values of all pixels within the ROI. For each tumour line, as well as for healthy brain regions, the average and standard deviation was calculated from all ROIs. All algorithms were implemented in Matlab .For each animal, pixel-by-pixel Δ165 lesions were highlighted by staining for the endothelial marker CD34 , glut-1  and the pericyte marker α-smooth muscle actin  according to previously published protocols . The macrovascular blood volume map of Mel57-VEGF-A165 tumours is shown in R2* was used in the tumour areas (region inside the box). Outside the tumour region, a low cutoff threshold for ΔR2* is used to maximise the contrast between striatum  and cortex  originates in spin-echo images following USPIO administration  has the advantage over Gd-DTPA (R1 relaxivity ∼5 mM−1 s−1) of potentially improving both (i) and (ii).In the current study, all tumour models displayed vessel leakage, albeit at different degrees. Leakage of Gd-DTPA from tumour vessels into the interstitium is however not restricted to the tumour interstitium but can extent into the surrounding normal tissue. This obviously precludes an accurate estimation of tumour edges. As tumour (and edge) detection rely on factors that include (i) the signal intensity contrast between the lesion and peritumoral, non-neoplastic tissue and (ii) the spatial resolution of the MR image, USPIO (R2 relaxivity ∼100 mR2* values are highly sensitive to small changes in regional blood volume. Thus USPIO imaging may be a very attractive alternative to Gd-DTPA imaging and will at least have added value, especially for detection and delineation of diffuse infiltrative brain tumours.In conclusion, detailed characterisation of the tumour vasculature by imaging vascular leakage and vascular volume provides a better understanding of the complex mechanisms associated with tumour development. Gadolinium-DTPA and USPIO contrast-enhanced MRI allow not only the detection of tumours but also characterisation of their vasculature, which is essential for diagnostic purposes and to assess efficacy of, for example, antiangiogenic therapy. Tumours from the three tumour models displayed three distinctive vascular morphologies, which were best assessed on post-USPIO gradient-echo images. The results of this study indicate that USPIO-induced Δ


The effect of 5-fluorouracil (5-FU) on the growth and cellular proliferation of hepatoma 3924A was studied using the following parameters as indices of tumour response: (1) volume measurements, (2) cell kinetic analysis including estimates of both growth and cell loss fractions, (3) changes in tumour histology and (4) tumour DNA content and DNA synthesis. Of a series of single intraperitoneally injected doses (25-300 mg/kg body weight), 150 mg/kg interrupted tumour growth most effectively with minimal toxicity within 168 h, and after 10 days treated tumour volumes were only 42% of untreated tumour size. Doses of 25 mg/kg failed to change the rate of growth while 300 mg/kg exceeded the LD50. Alterations of both tumour cell proliferation and histology developed well in advance of changes observed in growth. A dose of 150 mg/kg body weight blocked the transition of cells from G1 through S for a 24 h interval when cell kinetics were measured by 3H-TdR autoradiography. However, 3H-UdR incorporation into DNA following 5-FU suggested that cellular recovery from the drug was delayed for an additional 24 h. Concurrently, significant losses of tumour tissue and tumour DNA occurred during the first 48 h with an expected increase in both necrotic and connective tissue. During the subsequent 120 h both tumour and necrotic tissue had returned to non-treated levels, while kinetic analysis revealed (a) a slight reduction in the cell cycle time and growth fraction and (b) an increased cell loss factor. The observations from this tumour model system suggest that before using tumour volume or weight as an index of therapeutic response, the relationship between the kinetics of tumour cellularity and tumour volume must be defined.


In the present study we describe the effect of Zn(pic)2 and Zn(asp)2 on thereplication of VZV and on the infectivity of free virions. The experiments are done using BHK-21cells, a clinical isolate of VZV and Zn-complexes in concentration of 10 μM. When Zn-complexesare present during the whole period of infection, the yield of infectious virus progeny decreases upto 98%. The infectivity of VZV is completely restored after the removal of zinc. The virucidal effect ismanifested at the 2nd h of contact, when 90% of the virions are inactivated. The results show thatboth Zn(pic)2 and Zn(asp)2 specifically inactivate free VZV virions with no effect on viral replication.Zn(II) picolinate and aspartate, Zn(pic)


Primary chemotherapy in operable breast invasive carcinoma enables tumour reduction and conservative surgery. In order to search for one or more biological factors capable of predicting tumour behaviour under primary chemotherapy, and subsequent patient survival, an immunohistochemical study was performed with specific antibodies to p53, c-erbB-2 (Her-2/neu), Mib1 (antiKi-67), pS2, GST pi, oestrogen receptors (ERs) and progesterone receptors (PRs). Core biopsies, obtained before primary chemotherapy, were available from a series of 128 breast invasive carcinomas treated between January 1985 and April 1989, with a median follow-up of 93.3 months. Univariate statistical analysis showed that negative ER detection by immunohistochemistry (IHC) was highly correlated with chemosensitivity (P = 0.001). A high percentage of Mib1-positive tumour cells (> 40%), as well as initial tumour size less than 4 cm, were also correlated with tumour responsiveness to chemotherapy (P = 0.009 and P = 0.03). By multivariate analysis IHC-ER, Mib1 and initial tumour size were independent predictors, the last parameter being the most important. Concerning subsequent patient survival, c-erbB-2 overexpression, as detected by IHC, was significant with respect to overall survival (OS) (P = 0.0006), disease-free interval (DFI) (P = 0.03) and metastasis-free interval (MFI) (P = 0.008) by univariate analysis. Furthermore, c-erbB-2 was the major independent prognostic factor for OS and MFI by multivariate analysis.


In syngeneic mice, the H5V polyoma middle-T oncogene-transformed endothelioma cell line induces Kaposi's sarcoma-like cavernous haemangiomas that regress transiently, probably because of an anti-tumour immune response, but eventually grow progressively and kill the host. To evaluate the generation of tumour-specific cytotoxic T lymphocytes (CTLs), spleen cells of tumour-bearing mice were restimulated with irradiated H5V cells in mixed leucocyte-tumour cell cultures. Tumour-specific CTLs were demonstrable only when low numbers of H5V stimulator cells were used (<1 H5V cell per 50 splenocytes). We found that H5V cells secrete immunosuppressive mediators because CTL generation was blocked when H5V cells culture supernatants were added to allogeneic mixed leucocyte cultures. As numerous tumour-derived immunosuppressive mediators may interfere with interleukin 12 (IL-12) production, we tested whether IL-12 treatment of the tumour-bearing mice would augment their immune response and thus suppress tumour growth. Indeed, IL-12 inhibited tumour growth and prevented mortality, but did not increase anti-H5V CTL generation either in vitro or in vivo. Moreover, the anti-tumour activity in IL-12-treated mice was abrogated by anti-interferon (IFN)-gamma monoclonal antibody (MAb) co-administration. These results strongly suggest that the anti-tumour effect of IL-12 is principally mediated by IFN-gamma release that in turn blocks H5V cell proliferation and induces the release of factors that suppress angiogenesis.


Coumarin anticoagulants inhibit metastasis in several animal models, but the mechanism of this effect is uncertain. In order to determine the role of cytotoxic and/or cytostatic actions of coumarins on the tumour cells, we have studied the effects of warfarin on tumour cell growth in a model in which tumour metastasis is inhibited by this drug. Clonogenic assay, growth curve analysis and thymidine labelling index revealed that warfarin had no effects on Mtln3 mammary carcinoma cell growth in vitro at concentrations below 1 mM. The growth rate of subcutaneously implanted Mtln3 tumour deposits in female F344 rats, assessed by weight and by stathmokinetic analysis of the tumour tissue, was identical in warfarin-treated and control animals. Spontaneous metastasis from such tumours to the lungs was, however, significantly reduced in warfarin-treated animals . The mean plasma warfarin concentration in warfarin treated rats was 1.63 microM. These results suggest that warfarin treatment of the host animal can inhibit tumour metastasis without having any direct or indirect effect on the growth rate of the tumour cells.


The effects of the host's immune response on metastatic spread was investigated by observing the numbers of pulmonary metastases that developed from an s.c. implant of the Lewis lung carcinoma in C57BL mice in which different cell populations had been suppressed. Macrophage function was impaired by treatment with silica (Si), cortisone acetate (CA), or trypan blue (TB). T-cell function was depressed by adult thymectomy and sublethal irradiation, or by treatment with antilymphocyte serum (ALS). Metastasis was significantly increased and phagocytic activity decreased by Si and CA, but were unaffected by TB. Thymectomy and irradiation had no effect on metastases, whereas ALS when given before, but not after tumour growth, reduced their number. The antimetastatic action of the immunopotentiating agent C. parvum was investigated in these immunologically impaired mice. It was unaffected by Si, CA or TB. However, the inhibiting effect of these agents on phagocytic activity was overcome by treatment with C. parvum. Its antimetastatic action was unaffected in mice which had been thymectomized and irradiated, but could be abrogated by ALS. However, ALS was only able to prevent this activity if given before tumour growth; it was ineffective if given after tumour growth. This study showed that metastatic spread was inversely related to phagocytic activity. The antimetastatic effect of C. parvum appears to be mediated through macrophages in concert with a subpopulation of T lymphocytes, which were considered to be necessary in the sensitization arm of the response as opposed to the effector arm of this response.


Obstructive jaundice is most commonly due to luminal stones or lesions of the head of the pancreas and more rarely ampullary and primary common bile duct lesions. Obstruction due to lesions of the ampulla of Vater may be due to adenocarcinoma which has a significantly better long term prognosis than carcinomas located in the head of the pancreas. A case is presented where two tumours were identified at the ampulla of Vater of the resected specimen one an adenocarcinoma and the other a carcinoid tumour representing a collision tumour.


Over the last decade assessment of angiogenesis has emerged as a potentially useful biological prognostic and predictive factor in human solid tumours. With the development of highly specific endothelial markers that can be assessed in histological archival specimens, several quantitative studies have been performed in various solid tumours. The majority of published studies have shown a positive correlation between intra-tumoural microvessel density, a measure of tumour angiogenesis, and prognosis in solid tumours. A minority of studies have not demonstrated an association and this may be attributed to significant differences in the methodologies employed for sample selection, immunostaining techniques, vessel counting and statistical analysis, although a number of biological differences may account for the discrepancy. In this review we evaluate the quantification of angiogenesis by immunohistochemistry, the relationship between tumour vascularity and metastasis, and the clinicopathological studies correlating intra-tumoral microvessel density with prognosis and response to anti-cancer therapy. In view of the extensive nature of this retrospective body of data, comparative studies are needed to identify the optimum technique and endothelial antigens  but subsequently prospective studies that allocate treatment on the basis of microvessel density are required.British Journal of Cancer (2002) 86, 1566–1577. DOI: 10.1038/sj/bjc/6600315www.bjcancer.comCancer Research UK© 2002  Angiogenesis is the process in which new blood vessels arise from existing ones. This is present physiologically in the adult during wound healing, the development of the ovarian follicle and corpus luteum and in the proliferating endometrium. Pathological angiogenesis is also a component of much diverse pathology ranging from diabetes and atherosclerosis to cancer, a disease that cannot progress without the formation of new blood vessels .The measurement of angiogenesis is complicated by the fact that it is a dynamic process yet most studies to date have focused on the product of angiogenesis, the microvessel density, which was analysed at a particular point in time. Accepting this caveat there are extensive data in which the microvessel density correlates with metastasis and survival. Here, we discuss the relationship between angiogenesis and the intra-tumoural microvessel density (IMD), review the different techniques that have been used to measure IMD and then discuss the reasons why this relationship does not always hold.As angiogenesis is a dynamic process, comparisons with snapshot views of the tumour, as are seen in biopsy analyses, are not straightforward. On the other hand the only approaches that are available in the clinic for the serial measurement of tumour angiogenesis involve molecular imaging strategies, such as contrast-enhanced magnetic resonance imaging (MRI) and positron emission tomography (PET), that do not resolve data at the microscopic level. Thus it is important to assess the relationship between IMD and angiogenesis.Several studies have reported correlations between IMD and angiogenic growth factor expression, tumour growth and the occurrence of distant metastasis suggesting that IMD, as well as quantifying vascular density, reflects important information on the degree and function of tumour vasculature.Several clinicopathological studies have shown a direct association between angiogenic cytokine expression and IMD. Vascular endothelial growth factor (VEGF) expression has been shown to correlate with microvessel density in a number of solid tumours including prostate  has also been reported . The pro+ IMD and uPA/PAI-1 levels.Background extracellular matrix proteolysis is one of the most important steps in angiogenesis. The urokinase-type plasminogen activator system (uPAS) consisting of urokinase plasminogen activator (uPA), an extracellular proteolytic enzyme produced by tumour cells, its receptor uPA-R and their corresponding inhibitors plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2) are thought to play a major role in this process. uPA at the cell surface initiates a proteinase cascade leading to the breakdown of the extracellular matrix and thereby promoting cellular migration. The levels of uPA and its inhibitor PAI-1 are known prognostic factors in breast cancer. In a series of patients with breast cancer, uPA and PAI-1 contents were measured by ELISA in tissue extracts, in peripheral and central tumour tissue  , patient groups, therapies and data (vessel quantification) interpretation. The correct identification of the vascular hot spot within the tumour and observer experience are two of the most important factors. In one study that compared the effects of different methodologies on estimates of tumour vascularity, archival specimens of breast, lung and oral carcinoma, oral dysplasia and normal breast tissue were investigated. Pretreatment of sections , endothelial markers (vWF-von Willebrand factor and CD31 antibodies), method of quantification  and inter-observer variations were all found to alter the estimated vascularity and interestingly the treatment of sections before staining was the variable that most significantly altered the calculated vascularity of tumours  and OS (P=0.0029) in contrast to blood vessel counts using CD34 that did not correlate with RFS or OS (P=0.029) while CD34 determined IMD was not.The ability to distinguish quantitatively between tumour neovascularisation and pre-existing vessels may be important in the assessment of tumour angiogenesis and could provide more accurate prognostic information. This is now possible using markers for activated endothelium. These markers are suitable for proliferating endothelium giving none or poor staining of lymphatics and normal quiescent blood vessels. They mainly react with fresh or frozen tissues, their activity in paraffin-embedded specimens is fixation dependent. In a study of 106 patients with breast carcinoma, the IMD was assessed using a pan-endothelial marker CD34 and a monoclonal antibody to CD105 that preferentially reacts with endothelial cells in angiogenic tissues. IMD values for CD105 expression showed a statistically significant correlation with RFS . A low background staining and highly specific and intense labelling of endothelial cells is required. Once the vascular hot spot is defined, a higher magnification is selected in order to be able to count individual microvessels. Magnifications of the order of 200–400×and field sizes ranging from 0.12 to 1.00 mmAccording to an early study (2. The a-MVD is determined using the same grid and magnification (200×) as for h-MVD and calculating the mean of the vascular counts obtained in at least 10–15 random fields for each tissue section. Results for a-MVD are expressed as mean±standard deviation (vessels per mm2) (Quantification of stained vessels can be achieved by measuring highest microvascular density (h-MVD), the average microvessel density (a-MVD) or the microvascular volume (MVV). The area of highest microvascular density (the vascular hot spot) is located by scanning the section at 100×magnification. In practice, localisation of the highest density area normally involves counting up to three different areas. Three different fields are counted in each of these areas at 200×magnification, and the highest value taken as the h-MVD, expressed as vessels per mmr=0.7, P=0.00005). A significant reduction in OS was observed between patients stratified by Chalkley count in both univariate (P=0.02) and multivariate analysis (P=0.05). In another study in patients with node-positive breast carcinoma, Chalkley score was found to be the most significant independent predictor of outcome by multivariate analysis . Manual microvessel counting had no prognostic impact. In another study of 104 malignant mesotheliomas, IMD as assessed by Chalkley counting was assessed with respect to other known prognostic factors in malignant mesothelioma. Chalkley microvessel count was shown to be an independent prognostic factor in multivariate analysis (P=0.006) .As tumour metastasis is the major cause of mortality in cancer patients, a classification of the metastatic potential of a tumour could be of great clinical significance. A quantitative relationship between IMD, the number of intravascular tumour cells and the occurrence of pulmonary metastasis was shown in an animal tumour model more than 20 years ago (P=0.025). P=0.029). In another series of patients with node-negative breast cancer, assessment of IMD by immunohistochemistry with anti-CD31 antibody, IMD correlated significantly with recurrence in viscera, bone and soft tissue deposits  and the expression level of bFGF expression (P=0.045) were independent predictors for the development of metastases.The expression levels of genes that regulate metastasis and angiogenesis can predict metastatic potential in individual patients. This was demonstrated in a study of 46 renal cancers  . All patients with breast carcinomas containing more than 100 microvessels per 200×field experienced tumour recurrence within 33 months of diagnosis, compared with less than 5% of patients with breast cancer having 33 or fewer microvessels per 200×field. Moreover IMD was the only statistically significant predictor of OS in node-negative patients (P<0.001). In the study by 2 was significantly associated with node metastasis (P<0.00001). Vascularity was correlated with the size of the primary tumour and with poor differentiation. Within each subgroup of size or differentiation, tumours without nodal involvement had much lower IMD. Even with the short follow up in this study, high vascular counts correlated with early deaths.If IMD is associated with metastasis then it is logical to predict that IMD will also be associated with survival and this has been evaluated in most solid tumours including breast, ovary, bladder, head and neck and prostate cancers amongst others , tumour size and regional lymph node status retained independent prognostic value with respect to overall survival. Among these variables, tumour microvessel count considered as a continuous variable was the most important . Anti-CD34 antibody was used to measure angiogenesis in tumour samples. In multivariate analysis IMD, tumour microvessel count  were characterised by an absence of stroma and lack of new vessel formation (A further mechanism by which the discrepancy between IMD and survival may occur is vasculogenic mimicry, which has been described in melanoma. In aggressive primary and metastatic melanomas, the tumour cells generate microcirculatory channels composed of extracellular matrix that are lined by tumour cells (Despite the fact that the overall trend in most studies is that the assessment of IMD retains a prognostic value in the majority of solid tumours, no definitive conclusions can be drawn at present on the real clinical usefulness of this approach. Before adopting IMD as a prognostic marker in routine clinical application, appropriate prospective trials are needed to validate the results observed in retrospective studies. Furthermore the method of assessment of IMD is inconsistent and needs to be standardised and made more objective.There is sufficient evidence to justify the determination of IMD as a measure of the angiogenic activity of human solid tumours. This method may be improved by employing more selective and specific markers for activated/proliferating endothelium, improving staining techniques, using more objective and reproducible methods for microvessel counting and standardisation of the criteria for evaluation of IMD and of the identification of the neovascular hot spot within each tumour.As alternative methods like serum and tissue sampling of angiogenic peptides, measurement of endothelial cell proliferation, cell adhesion molecules and proteolytic enzymes as well as dynamic contrast enhanced MRI become available, these may enhance the accuracy of the measurement of angiogenic activity in solid tumours.A strong correlation has been confirmed between the IMD of a primary tumour and its potential to develop locoregional or distant metastasis. Assessment of tumour vascularity is now an established prognostic indicator in early stage breast cancer, NSCLC and prostatic carcinoma . In thesCertain issues remain unresolved. Besides the technical standardisation issues, it is unclear whether antibodies directed against activated endothelium will predict metastasis and survival better than other pan-endothelial methods. This issue needs to be addressed in a large study before we can establish prospective trials that are ultimately needed to validate IMD as a guide to therapy.


The biodistribution of two recently developed tumour markers, trimethylated (CP(Me)3) and trimethoxylated (CP(OMe)3) carotenoporphyrin, was investigated by means of laser-induced fluorescence (LIF) after i.v. injection into 38 tumour-bearing (MS-2 fibrosarcoma) female Balb/c mice. At 3, 24, 48 or 96 h after administration, the carotenoporphyrin fluorescence was measured in tumoral and peritumoral tissue, as well as in the abdominal, thoracic and cranial cavities. The fluorescence was induced by a nitrogen laser-pumped dye laser, emitting light at 425 nm, and analysed by a polychromator equipped with an image-intensified CCD camera. The fluorescence was evaluated at 490, 655 and 720 nm: the second and third wavelengths represent the carotenoporphyrin (CP)-related peaks, whereas the first one is close to the peak of the tissue autofluorescence. The tumour and the liver were the two tissue types showing the strongest carotenoporphyrin-related fluorescence, whereas the cerebral cortex and muscle consistently exhibited weak substance-related fluorescence. In most tissue types, the fluorescence intensities decreased over time. A few exceptions were observed, notably the liver, in which the intensity remained remarkably constant over the time period investigated.


A direct relationship between amount of EA and tumour growing capacity was found. EA were detected also on fast dividing testicular cells. It is suggested that EA expression on tumour cells is related to a cell membrane function controlling mitosis rather than to a function specifically related to the neoplastic status. Tumour take of low doses of 2 EA-bearing sarcomata was found to be enhanced in anti-embryo immune BALB/c mice in comparison with that in normal and anti-fibroblast immune mice.The amount of embryonic antigens (EA) was estimated in 13 BALB/c fibrosarcomata by