A comparative study of tissue distribution and photodynamic therapy selectivity of chlorin e6, Photofrin II and ALA-induced protoporphyrin IX in a colon carcinoma model.

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 (0.6 J cm-2 per pulse, 1 Hz). 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 (50, 80 and 100%) 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.     

Selective accumulation of ALA-induced PpIX and photodynamic effect in chemically induced hepatocellular carcinoma

The possibility of 5-aminolaevulinic acid-based photodynamic therapy (ALA-PDT) for liver cancer was investigated using a chemically induced hepatocellular carcinoma (HCC) model. Endogenously synthesised protoporphyrin IX (PpIX) following the administration of ALA is an effective photosensitiser for PDT. We determined the fluorescence intensity of PpIX in HCC and nontumoral tissue in the liver. 5-Aminolaevulinic acid was intravenously injected to male Fisher rats with HCC at a dose of 500 mg x kg(-1), and the fluorescence intensity in each tissue sample excised from liver was measured with a spectrofluorometer at 1, 3 and 6 h after administration. Fluorescence intensity was at a peak of 3 h after administration in both HCC and nontumoral tissue. The accumulation of PpIX in HCC was higher than that in the nontumoral tissue at 1 h (P<0.001) and 3 h (P<0.05) after ALA administration. Based on these results, PDT was performed on HCC at 3 h after 500 mg x kg(-1) ALA administration before laser irradiation of 30 J per tumour. Antitumour effect was more evident in HCC than in the nontumoral tissue surrounding HCC. These findings suggest the possibility to detect HCC by fluorescence and to treat HCC by light.     

Adjuvant intraoperative photodynamic therapy diminishes the rate of local recurrence in a rat mammary tumour model.

The use of photodynamic therapy (PDT) as an adjunct to curative tumour resection was investigated in a tumour recurrence model, using rat mammary adenocarcinoma BN472. Tumours were inoculated subcutaneously in 60 animals and resected after 21 days of growth. Immediately after removal, the operation site was exposed to 320-450 nm light of 0.1 W cm-2 and 60 J cm-2 after photosensitisation with either Photofrin (5 mg kg-1 i.v. 48 h before illumination) or 5-aminolaevulinic acid (ALA) (2 mg ml-1 in drinking water for 9 days). Porphyrin concentrations were measured in tissue samples. After 28 days, animals treated with adjunctive PDT had a significantly longer tumour-free interval than controls (P < 0.01); median 25 days (Photofrin), 18 days (ALA), 14 days (controls). Moreover, in the PDT groups significantly fewer rats had lymph node metastasis. A prophyrin concentration ratio between tumour and mammary tissue of 2:1 was found after Photofrin and 4:1 after ALA. The results indicate that adjuvant intraoperative PDT may be a safe and effective method of destroying residual tumour, thereby preventing locoregional tumour recurrence.     

Evaluation of tumour and tissue distribution of porphyrins for use in photodynamic therapy.

A range of pure, monomeric porphyrins were synthesised and their localising capacities compared to HpD and Hp at 6 h and 24 h post injection in the mouse C6 intracerebral glioma model as well as in normal brain, skin, muscle, kidney, spleen, liver, lung and whole blood. The partition coefficients were examined between PBS and 2-octanol over the pH range 7.4-6.6 and pH profiles were established. A parabolic relationship was observed between log (porphyrin tumour concentration) at pH 7.4, with maximal tumour localisation at log (partition coefficient), pi, of approximately zero. Porphyrins with side chains with nett cationic character also exhibited up upward (parabolic) dependence on pi for most tissues studied, with maximal porphyrin localisation at pi of 0-0.5. In contrast, those porphyrins with nett anionic character exhibited a downward (negative) parabolic trend for all eight tissues studied, with minimal porphyrin localisation at pi of approximately zero. Four porphyrins (4, 11, 12, 13) exhibited similar or better tumour localisation than HpD, and two (11 and 12) offer promise as lead compounds for the design of improved porphyrins for use in PDT.     

ALA- and ALA-hexylester-induced protoporphyrin IX fluorescence and distribution in multicell tumour spheroids

Synthesis of protoporphyrin IX (PpIX) in intact murine mammary cancer cell spheroids is reported from optical sections obtained using a laser scanning confocal fluorescence microscope. EMT6 spheroids 275-350 microm in diameter were incubated in 0.1-15 mM aminolevulinic acid (ALA) or 0.001-2 mM ALA-hexylester (h-ALA) to test the ability of both pro-drugs to diffuse into the spheroids and induce PpIX production. Spheroids incubated with ALA show significant fluorescence nonuniformity for all concentrations, with the outermost cells exhibiting greater porphyrin fluorescence. Comparable levels of fluorescence throughout the optical section are achieved with approximately 100-fold lower h-ALA concentrations, indicating that the interior cells maintain esterase activity and porphyrin synthesis and that h-ALA diffuses efficiently to the spheroid interior. Fluorescence gradients are less pronounced with h-ALA incubation, in part because of apparent saturation of esterase activity in the spheroid perimeter. Proliferating (Ki67 positive) and quiescent cell populations exhibit remarkably different h-ALA concentration dependencies. The incubation concentration resulting in maximum fluorescence with ALA is 10 mM, while the optimal concentration for h-ALA is 200-fold lower at 0.05 mM. Exceeding these optimal concentrations for both pro-drugs leads to an overall loss of fluorescence.     

Interstitial photodynamic therapy in a rat liver metastasis model.

Photodynamic therapy (PDT) of hepatic tumours has been restricted owing to the preferential retention of photosensitizers in liver tissue. We therefore investigated interstitial tumour illumination as a means of selective PDT. A piece of colon carcinoma CC531 was implanted in the liver of Wag/Rij rats. Photofrin was administered (5 mg kg-1 i.v.) 2 days before laser illumination. Tumours with a mean (+/- s.e.) diameter of 5.7 +/- 0.1 mm (n = 106, 20 days after implantation) were illuminated with 625 nm light, at 200 mW cm-1 from a 0.5 cm cylindrical diffuser and either 100, 200, 400, 800 or 1600 J cm-1. Control groups received either laser illumination only, Photofrin only or diffuser insertion only. Short-term effects were studied on the second day after illumination by light microscopy and computer-assisted integration of the circumference of damaged areas. Long-term effects were studied on day 36. To determine the biochemistry of liver damage and function, serum ASAT and ALAT levels were measured on day 1 and 2, and antipyrine clearance on day 1. Tumour and surrounding liver necrosis increased with light dose delivered (P < 0.001). Best long-term results were obtained at 800 J cm-1 with complete tumour remission in 4 out of 6 animals. No deterioration in liver function was found. The results of this study show the ability of interstitial PDT to cause major destruction of tumour tissue in the liver combined with minimal liver damage.     

Photobleaching during and re-appearance after photodynamic therapy of topical ALA-induced fluorescence in UVB-treated mouse skin

Photodynamic therapy (PDT) using protoporphyrin IX (PpIX) induced by topically applied 5-aminolevulinic acid (ALA) seems a promising alternative for the treatment of superficial non-melanoma skin cancer and actinic keratosis. In this study, the kinetics of new PpIX fluorescence arising after a PDT treatment that had photobleached the original fluorescence were determined. Our purpose was to examine the feasibility of multiple irradiations, following a single topical ALA application, to increase PDT efficacy. In addition, photobleaching during PDT and the fluorescence spectra during and after PDT were studied. As a model we used hairless mice with and without UVB-induced skin lesions. ALA was applied to the skin for 4 hr. An illumination was delivered either immediately after application or 6 hr after the end of the application (at interval of maximum fluorescence). During PDT, the fluorescence of normal skin decreased at a faster rate than the fluorescence of the skin lesions. In the fluorescence study after PDT, the areas treated immediately post-application showed a fluorescence increase over time similar to that in non-treated areas on the same mice. A remarkable result was that the fluorescence of areas treated at maximum fluorescence increased, whereas the fluorescence of non-treated areas did not increase over time. With both treatment intervals the new fluorescence showed a characteristic PpIX spectrum. Our results demonstrate that a second illumination, when new PpIX fluorescence has been formed, may increase PDT efficacy after topical ALA application. This finding has been demonstrated previously for systemic ALA administration. Int. J. Cancer 72:110–118, 1997. © 1997 Wiley-Liss Inc.     

表皮生长因子增强二甲基肼诱发大白鼠大肠癌的效力

 本文应用动物诱癌试验观察内源性和外源性表皮生长因子（EGF）对二甲基肼（DMH）诱发WISTAR鼠大肠癌效力的影响。将60只大白鼠随机分咸四组：高EGF组、普通组（对照组）、低EGF组、单EGF组、用DMH连续诱癌20周。结果发现高EGF组动物诱癌阳性率、平均负瘤数、肿瘤平均体积均明显高于普通组，更高于低EGF组（P＜0．05），单EGF组无肿瘤产生。提示EGF可增强DMH诱发WISTRR鼠大肠癌的效力，但不能独立致癌，在本实验中EGF有类似促癌剂的作用。     

Correlation of distribution of sulphonated aluminium phthalocyanines with their photodynamic effect in tumour and skin of mice bearing CaD2 mammary carcinoma.

A chemical extraction assay and fluorescence microscopy incorporating a light-sensitive thermoelectrically cooled charge-coupled device (CCD) camera was used to study the kinetics of uptake, retention and localisation of disulphonated aluminium phthalocyanine (A1PcS2) and tetrasulphonated aluminium phthalocyanine (A1PcS4) at different time intervals after an i.p. injection at a dose of 10 mg kg-1 body weight (b.w.) in tumour and surrounding normal skin and muscle of female C3D2/F1 mice bearing CaD2 mammary carcinoma. Moreover, the photodynamic effect on the tumour and normal skin using sulphonated aluminium phthalocyanines (A1PcS1, A1PcS2, A1pcS4) and Photofrin was compared with respect to dye, dye dose and time interval between dye administration and light exposure. The maximal concentrations of A1PcS2 in the tumour tissue were reached 2-24 h after injection of the dye, while the amounts of A1PcS4 peaked 1-2 h after the dye administration. A1PcS2 was simultaneously localised in the interstitium and in the neoplastic cells of the tumour, whereas A1PcS4 appeared to localise only in the stroma of the tumour. The photodynamic efficiency (light was applied 24 h after dye injection at a dose of 10 mg kg-1 b.w.) of the tumours was found to decrease in the following order: A1PcS2 > A1PcS4 > Photofrin > A1PcS1. Furthermore, photodynamic efficacy was strongly dependent upon dye doses and time intervals between dye administration and light exposure: the higher the dose, the higher the photodynamic efficiency. The most efficient photodynamic therapy (PDT) of the tumour was reached (day 20 tumour-free) when light exposure took place 2 h after injection of A1PcS2 (10 mg kg-1). A dual intratumoral localisation pattern of the dye, as found for A1PcS2, seems desirable to obtain a high photodynamic efficiency. The kinetic patterns of uptake, retention and localisation of A1PcS2 and A1PcS4 are roughly correlated with their photodynamic effect on the tumour and normal skin.     

Intra-luminal tumour cells and peri-anastomotic tumour growth in experimental colonic surgery.

Most loco-regional tumour recurrences following colorectal cancer surgery are extra-luminal. This study was designed to determine the conditions necessary for viable intra-luminal tumour cells to give rise to extra-luminal tumour growth in an experimental animal model. When carcinoma cells were injected into the colonic lumen in the absence of a colonic wound, tumour growth was rare (1 of 24 animals). However, when tumour cells were injected intra-luminally either in the presence of an apparently watertight colotomy repair or prior to the transmural insertion of interrupted sutures, peri-anastomotic +/- widespread peritoneal tumour growth was consistently observed. Tumour growth did not occur on the mucosa of the bowel. These findings suggest that viable intra-luminal tumour cells can migrate across a clinically intact experimental large bowel anastomosis or along transmural suture tracts to give rise to extra-luminal tumour growth.     

Toremifene resistance in a rat mammary tumour model.

The effect of Toremifene on cell growth in vitro was tested on the R3230AC rat mammary adenocarcinoma as model. Toremifene differed from Tamoxifen in that it did not induce resistance; some cross-resistance was observed in Tamoxifen tolerant tumour cell lines. Toremifene does not influence P-glycoprotein expression in this model and at the levels studied.     

The effect of localized porphyrin photodynamic therapy on the induction of tumour metastasis

Studies were performed to determine whether localized treatment of subcutaneously growing Lewis Lung Carcinoma (LLC) in C57BL/6 mice with porphyrin photodynamic therapy (PDT) affects the formation of distant metastases. Treatments consisted of a 10 mg kg-1 dose of dihematoporphyrin-ether (Photofrin II) followed 24 h later by local tumour irradiation with 630 nm red light. Total doses of light ranged from 0-500 J cm-2 and the irradiance of delivered light was 150 mW cm-2. Primary LLC tumours were treated at a volume of 25-30 mm3, and lung metastases were determined 21 days following transplantation. Mice exposed to PDT treatment which produced either partial or complete local tumoricidal responses had significantly decreased numbers of metastatic lung colonies compared to controls. In addition, PDT treated mice had equal or less metastatic lung colonies than comparable mice treated with local surgical excision of the primary LLC lesion. These results indicate that local PDT does not enhance metastatic spread of LLC following either curative or noncurative treatments.     

The effect of therapeutic drugs used in inflammatory bowel disease on the incidence and growth of colonic cancer in the dimethylhydrazine rat model.

An increased incidence of colonic cancer is associated with chronic inflammatory bowel disease. Sulphasalazine, metronidazole and more recently, modified forms of 5-aminosalicylic acid are used for maintenance therapy of inflammatory bowel disease. In a series of experiments, we used the 1,2-dimethylhydrazine animal model of colonic cancer in conjunction with these drugs, to study the effect on the development of colon cancer. Inbred male Wistar rats were divided into groups receiving orally: metronidazole 18 mg Kg-1 dy-1; sulphasalazine 60 mg Kg-1 dy-1; 5-aminosalicylic acid 30 and 60 mg Kg-1 dy-1 and olsalazine 60 mg Kg-1 dy-1 administered daily. Half of each group also received weekly injections of DMH 40 mg Kg-1. Metronidazole, sulphasalazine and 30 mg Kg-1 dy-1 5-aminosalicylic acid were co-carcinogenic, increasing either the number of cancers or tumour size. In contrast 60 mg Kg-1 dy-1 5-aminosalicylic acid inhibited tumour size and olsalazine had no effect. These results may have a bearing on long term maintenance therapy in inflammatory bowel disease.     

The effect of serum CEA on the distribution and clearance of anti-CEA antibody in a pancreatic tumour xenograft model

A human pancreatic adenocarcinoma was used to develop two histologically distinct xenograft lines, one associated with high levels (180-2000 ng ml-1) and one with low levels (greater than 2.0 less than 8.0 ng ml-1) of serum carcinoembryonic antigen (CEA). A strong correlation was found between tumour size and both circulating and tumour CEA levels in the former group, and also correlation at the 5% level between tumour size and serum CEA in the latter. Administration of either monoclonal or polyclonal 125I-anti-CEA antibody led to the formation of intravascular antigen-antibody immune complexes in mice with high CEA levels, and these were rapidly cleared by the liver, deiodination commencing within the first hour. Blood activity was reduced to 20% of the injected dose by 15 min, and by 24 h the radioactivity in all tissues except muscle was significantly below that found in either the low CEA group or in mice without tumours. No difference in radio-antibody clearance pattern was found between mice without tumours and the group with low levels of serum CEA. In spite of higher levels of CEA within the tumour in mice with elevated serum CEA, the rapid clearance of antigen-antibody complexes reduced tumour localisation to one quarter of that seen in mice with low serum, and correspondingly low tumour, CEA levels. Gamma-camera imaging confirmed these results. Possible implications to patient selection and treatment are discussed.     

The effect of tumour size on ferromagnetic embolization hyperthermia in a rabbit liver tumour model.

OBJECTIVE: Ferromagnetic Embolization Hyperthermia (FEH) consists of arterially embolizing tumours with ferromagnetic particles to cause hysteretic heating upon subsequent exposure to an alternating magnetic field. The objective was to determine the effect of tumour size during FEH using a rabbit liver tumour model. METHOD: Thirty-three rabbits containing implanted hepatic VX2 carcinomas received a hepatic arterial infusion of ferromagnetic particles suspended in lipiodol. Following hysteretic heating, tumour and normal hepatic tissues were chemically analysed for iron content. Tumours were classed as small if their mass was less than the median mass for the whole group of subjects (2.1 g), and as large if their mass was greater than or equal to the median. To control for variability in tumour iron concentration, 13 small tumours were matched to 13 large tumours by iron concentration, and their heating characteristics compared. RESULTS: The heating rate in large tumours (median = 5.0 degrees C/min) was significantly greater than that in the matched small tumours (median = 2.8 degrees C/min), p = 0.006. Regression analysis determined that the slope of the heating rate vs iron concentration curve for large tumours was 1.5 times greater than that for the matched small tumours, p < 0.001. After cessation of heating in large tumours, there was continued heat dissipation into surrounding tissues, which led to anomalous temperature increases. There was an inverse linear relationship between tumour size and tumour iron concentration for a given dose of particles. CONCLUSION: For a given tumour iron concentration, larger tumours heat at a greater rate than small tumours, due to the poorer tissue cooling and better heat conduction in the necrotic regions of large tumours. This warrants further investigation as this finding could confer a significant advantage on FEH over other hyperthermic modalities in the treatment of hepatic malignancies.     

Effects of epidermal growth factor and mitogenic blockers on rat colonic tumour growth in vivo

A protocol for tumour cell inoculation (Per192NR and Per237 rat colon cancer cells) and implantation of mini-osmotic pumps in inbred rats was established. Surgical procedures and tumour growth did not affect red and white blood cell counts. Per237 did not grow in these rats while Per192NR formed poorly differentiated carcinomas which were not affected by EGF intraperitoneal (IP) or intravenous (IV), oleic acid (IP) or verapamil (IP or oral) administration. Haematological parameters and rat growth rates were not affected by EGF (IP and IV), verapamil (IP) and oleic acid (IP) treatment. This protocol provides a method for studying the effect of various cancer drugs in vivo.     

Lipogenesis in tumour and host tissues in mice bearing colonic adenocarcinomas.

Although animals bearing the MAC16 colon adenocarcinoma showed progressive weight loss, the average food consumption (15.1 +/- 0.6 Kcal day-1) did not differ from non tumour-bearing controls (15.3 +/- 0.3 Kcal day-1), while animals bearing a related colon adenocarcinoma, MAC13, which had no effect on body weight had a significantly (P less than 0.01) elevated food intake (16.4 +/- 0.3 Kcal day-1) above controls. Weight loss in animals bearing the MAC16 tumour was associated with a significant reduction in the percentage contribution of the kidneys, colon and epididymal fat pads to the total body weight. Although loss of body fat occurred only in the MAC16 model, both tumours were capable of synthesising lipids from glucose both in vitro and in vivo at the same rate. In addition both tumours increased the rate of lipogenesis from glucose in kidney, liver and epididymal fat pads of the host. Lipogenesis from glucose would be expected to result in a loss of utilisable carbohydrate energy and thus would be expected to increase the overall energy requirements in the tumour-bearing state leading to catabolism of host body tissues if the energy intake is not increased.     

Enhancement of interstitial photodynamic therapy by mitomycin C and EO9 in a mouse tumour model

The tumoricidal effect of interstitial photodynamic therapy (IPDT) using Photofrin was found to increase when combined with the bioreductive alkylating agent mitomycin C (MMC) and, to a lesser extent, with the indoloquinone EO9. When MMC was given prior to IPDT of RIFI tumours, the light dose required for a given regrowth time or for 50% cure was reduced by a factor of 2 compared with IPDT alone. MMC given immediately after illumination did not increase the effects of IPDT, although MMC plus illumination without photosensitizer produced a significant increase in regrowth time compared with MMC or light alone. Combination of IPDT with EO9, given directly before illumination, only marginally increased the tumour regrowth times at non-toxic doses. These results demonstrate that combining IPDT with MMC greatly improves the tumour response. Factors such as PDT-induced hypoxia, pH changes, temperature in-creases and production of toxic reactive oxygen species by both drugs may play a role in the enhanced MMC cvtotoxicity.     

Enhancing the photodynamic effect of hypericin in tumour spheroids by fractionated light delivery in combination with hyperoxygenation

The aim of this study was to explore the hypothesis of oxygen depletion during light irradiation as a possible explanation for the incomplete response seen after hypericin-mediated photodynamic therapy (PDT) under specific conditions. To investigate this, we performed PDT experiments using transitional cell carcinoma spheroids with fractionated light irradiation and hyperoxygenation. After 2-h incubation with 3 different hypericin concentrations, spheroids were irradiated either continuously or with fractionated light delivery. The effect of hyperoxygenation was investigated by bubbling normobaric oxygen in the solution surrounding the spheroids before continuous irradiation or during the dark interval of light fractionation. The PDT efficacy was evaluated with an MTT antiproliferation assay and apoptotic cells were visualized after PDT by DAPI staining. Our results show that fractionated light delivery with dark intervals ranging from 1 to 10 min does not enhance the PDT efficacy in spheroids at all, whereas hyperoxygenation, using appropriate hypericin concentrations and oxygenation intervals, results in a virtually complete malignant cell killing through apoptosis. This study suggests that oxygen depletion is the major source of relative treatment failure in hypericin-mediated PDT with spheroids, which can only be overcome with hyperoxygenation. Therefore, whole bladder wall PDT with hypericin is likely to become a very efficient antitumoural treatment against superficial bladder cancer, on the condition that instillation fluids are hyperoxygenated during light irradiation.     

Hyperthermia improves the antitumour effect of metronomic cyclophosphamide in a rat transplantable brain tumour.

BACKGROUND AND PURPOSE: As low-dose metronomic cyclophosphamide (CTX) and hyperthermia (HT) both exert antitumour effects in part through antiangiogenic mechanisms, interactive effects of the two modalities were explored. MATERIALS AND METHODS: Subcutaneously implanted rat tumours (BT4An) were treated with CTX 35 mg/kg i.p. three doses a week for two weeks, local water-bath HT yielding mean tumour temperature of 43 degrees C for one hour at day 0, both modalities combined (CTX-HT(0)), or saline. TUNEL assays, immunohistochemical staining of thrombospondin 1 (TSP-1) and real time RT-PCR of TSP-1 mRNA were analysed the first three hours after completed treatment day 0. RESULTS: Metronomic dosed CTX (p=0.006) and HT (p<0.001) both delayed tumour growth. The combined regimen was superior to either modality alone (p<0.001). Complete tumour regressions were observed in CTX (6%), HT (12%), and CTX-HT(0) (41%) treated rats. TSP-1 protein was specifically upregulated in the vascular matrix of tumours receiving CTX (weak), HT (moderate) and CTX-HT(0) (strong). In contrast, reduced expression of TSP-1 protein was observed in tumour cells after HT alone and CTX-HT(0). TUNEL assays indicated induction of apoptosis by HT and CTX-HT(0) 90 minutes after end of the first treatment. CONCLUSION: A single session of local HT enhances the effects of low-dose metronomic CTX, possibly in part mediated through a differential effect on TSP-1 protein levels in tumour cells and tumour vasculature.