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Background: The integrin-binding protein osteopontin is strongly associated with tumour development, yet is an abundant dietary component as a constituent of human and bovine milk. Therefore, we tested the effect of orally administered osteopontin (o-OPN) on the development of subcutaneous tumours in mice., Methods: Bovine milk osteopontin was administered in drinking water to tumour-bearing immune-competent mice. Tumour growth, proliferation, necrosis, apoptosis and blood vessel size and number were measured. Expression of the α₉ integrin was determined., Results: o-OPN suppressed tumour growth, increased the extent of necrosis, and induced formation of abnormally large blood vessels. Anti-OPN reactivity detected in the plasma of OPN-null mice fed OPN suggested that tumour-blocking peptides were absorbed during digestion, but the o-OPN effect was likely distinct from that of an RGD peptide. Expression of the α₉ integrin was detected on both tumour cells and blood vessels. Potential active peptides from the α₉ binding site of OPN were identified by mass spectrometry following in vitro digestion, and injection of these peptides suppressed tumour growth., Conclusions: These results suggest that peptides derived from o-OPN are absorbed and interfere with tumour growth and normal vessel development. o-OPN-derived peptides that target the α₉ integrin are likely involved.

Background: Hypoxia-inducible factor-1 (HIF-1) mediates the transcriptional response to hypoxic stress, promoting tumour progression and survival. This study investigated the acute effects of the small-molecule HIF-pathway inhibitor NSC-134754., Methods: Human PC-3LN5 prostate cancer cells were treated with NSC-134754 for 24 h in hypoxia. Orthotopic prostate tumour-bearing mice were treated with a single dose of NSC-134754 for 6, 24 or 48 h. Treatment response was measured using magnetic resonance spectroscopy and imaging. Ex-vivo histological validation of imaging findings was also sought., Results: In vitro, NSC-134754 significantly reduced lactate production and glucose uptake (P<0.05), while significantly increasing intracellular glucose (P<0.01) and glutamine uptake/metabolism (P<0.05). Increased glutamine metabolism was independent of c-Myc, a factor also downregulated by NSC-134754. In vivo, a significantly higher tumour apparent diffusion coefficient was determined 24 h post-treatment (P<0.05), with significantly higher tumour necrosis after 48 h (P<0.05). NSC-134754-treated tumours revealed lower expression of HIF-1α and glucose transporter-1, at 6 and 24 h respectively, while a transient increase in tumour hypoxia was observed after 24 h. Vessel perfusion/flow and vascular endothelial growth factor levels were unchanged with treatment., Conclusion: NSC-134754 induces metabolic alterations in vitro and early anti-tumour activity in vivo, independent of changes in vascular function. Our data support the further evaluation of NSC-134754 as an anti-cancer agent.

Background: High vascular endothelial growth factor (VEGFA) levels at the time of diagnosis confer a worse prognosis to multiple malignancies. Our aim was to investigate the role of VEGFA in promoting tumour growth through interaction with its environment., Methods: HL-60 cells were transduced with VEGFA165 or control vector using retroviral constructs. Control cells (n=7) or VEGFA165 cells (n=7) were subcutaneously injected into NOD/SCID mice. Immunohistochemistry of markers for angiogenesis (CD31) and cell proliferation (Ki67) and gene expression profiling of tumours were performed. Paracrine effects were investigated by mouse-specific cytokine arrays., Results: In vivo we observed a twofold increase in tumour weight when VEGFA165 was overexpressed (P=0.001), combined with increased angiogenesis (P=0.002) and enhanced tumour cell proliferation (P=0.001). Gene expression profiling revealed human genes involved in TGF-β signalling differentially expressed between both tumour groups, that is, TGFBR2 and SMAD5 were lower expressed whereas the inhibitory SMAD7 was higher expressed with VEGFA165. An increased expression of mouse-derived cytokines IFNG and interleukin 7 was found in VEGFA165 tumours, both described to induce SMAD7 expression., Conclusion: These results suggest a role for VEGFA-driven tumour growth by TGF-β signalling inhibition via paracrine mechanisms in vivo, and underscore the importance of stromal interaction in the VEGFA-induced phenotype.

Background: The cancer stem cell hypothesis suggests that neoplastic clones are maintained exclusively by a small subpopulation of cells, which have indefinite proliferation and differentiation potentials and give rise to phenotypically diverse cancer cells. Cancer stem cells have been isolated by their ability to efflux Hoechst 33342 dye and are referred to as the 'side population' (SP)., Methods and Results: The Hoechst efflux assay was used to isolate and characterize the SP from murine D121 lung carcinoma cells. Here, we demonstrated that D121-SP cells contain cancer stem cell characteristics, that is, upregulation of the transcription factors SOX2 and Oct 4 in D121-SP cells. In addition, the migration of D121-SP was decreased, and apoptosis of D121-SP was upregulated following knocking down of SOX2 in D121 cells. Importantly, downregulation of SOX2 in D121 cells markedly suppressed their metastatic potential in syngeneic mice., Conclusions: These results suggest that the SP is an enriched source of lung tumour cells with stem cell properties and that SOX2 has an important role in maintaining stem cell properties and functions that may be a potential target for effective lung cancer therapy.

Background: To enhance the success rate of nanocarrier-mediated chemotherapy combined with an anti-angiogenic agent, it is crucial to identify parameters for tumour vasculature that can predict a response to the treatment of the anti-angiogenic agent., Methods: To apply transforming growth factor (TGF)-beta type I receptor (TbetaR-I) inhibitor, A-83-01, to combined therapy, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was carried out in mice bearing colon 26 cells using gadolinium (Gd)-DTPA and for its liposomal formulation to evaluate changes in tumour microvasculature following A-83-01. Tumour vascular parameters from DCE-MRI were compared with histological assessment and apparent diffusion coefficient of water in tumour generated by diffusion-weighted MRI., Results: Contrary to evaluations reported for anti-angiogenic agents, A-83-01 treatment increased the initial area under the Gd concentration-time curve (IAUGC60), volume transfer constant (K(trans)) and fractional plasma volume (v(p)) significantly within 24 h, that was positively related to alpha-smooth muscle actin-positive pericyte coverage and tumour cell proliferation, and was correlated inversely with the apparent diffusion coefficient. The vascular function of the tumour improved by A-83-01 treatment was well assessed on post-liposomal Gd-DTPA-enhanced MR images, which predicted delivery of a liposomal drug to the tumour., Conclusion: These findings suggest that DCE-MRI and, in particular, K(trans) and v(p) quantitation, provide important additional information about tumour vasculature by A-83-01 treatment.

Gastric cancer metastasised to the liver was found to overexpress HER2 at a significantly higher incidence than primary gastric cancers. The purpose of the present study was to investigate the possibility of molecular therapy targeting HER2 overexpression in gastric cancer liver metastasis. We developed three new HER2-overexpressing gastric cancer cell lines (GLM-1, GLM-2, GLM-4) without epidermal growth factor receptor (EGFR) mutations derived from such liver metastasis, two of which had HER2 gene amplifications. All these GLM series of cell lines were highly sensitive to gefitinib in vitro, a specific inhibitor of EGFR tyrosine kinase (Iressa) rather than anti-HER2 antibody trastuzumab (Herceptin), whereas most of the HER2 low-expressing counterparts were not. In these HER2-overexpressing GLM series, protein kinase B (Akt), but not extracellular signal-regulated kinase 1/2 (ERK1/2), was constitutively phosphorylated, and gefitinib efficiently inhibited this Akt phosphorylation, induced strong apoptosis in vitro and exhibited antitumour activity in tumour xenografts in nude mice. This gefitinib-mediated antitumour effect in xenograft was significantly potentiated by trastuzumab treatment. On the other hand, gefitinib-resistant cells (GLM-1R) exhibited increased EGFR expression, followed by constitutive activation of mitogen-activated protein kinase (MAPK) pathway. These results suggest that the antitumour effect of gefitinib is due to the effective inhibition of HER2-driven constitutive activation of phosphatidylinositol-3-kinase (PI3K)/Akt pathway, and that the acquired resistance to gefitinib is due to the constitutive activation of Ras/MAPK pathway in compensation for PI3K/Akt pathway. Gastric cancer liver metastasis with HER2 overexpression would be a potential molecular target for gefitinib and trastuzumab.

Muscle protein degradation is thought to play a major role in muscle atrophy in cancer cachexia. To investigate the importance of the ubiquitin-proteasome pathway, which has been suggested to be the main degradative pathway mediating progressive protein loss in cachexia, the expression of mRNA for proteasome subunits C2 and C5 as well as the ubiquitin-conjugating enzyme, E2(14k), has been determined in gastrocnemius and pectoral muscles of mice bearing the MAC16 adenocarcinoma, using competitive quantitative reverse transcriptase polymerase chain reaction. Protein levels of proteasome subunits and E2(14k) were determined by immunoblotting, to ensure changes in mRNA were reflected in changes in protein expression. Muscle weights correlated linearly with weight loss during the course of the study. There was a good correlation between expression of C2 and E2(14k) mRNA and protein levels in gastrocnemius muscle with increases of 6-8-fold for C2 and two-fold for E2(14k) between 12 and 20% weight loss, followed by a decrease in expression at weight losses of 25-27%, although loss of muscle protein continued. In contrast, expression of C5 mRNA only increased two-fold and was elevated similarly at all weight losses between 7.5 and 27%. Both proteasome functional activity, and proteasome-specific tyrosine release as a measure of total protein degradation was also maximal at 18-20% weight loss and decreased at higher weight loss. Proteasome expression in pectoral muscle followed a different pattern with increases in C2 and C5 and E2(14k) mRNA only being seen at weight losses above 17%, although muscle loss increased progressively with increasing weight loss. These results suggest that activation of the ubiquitin-proteasome pathway plays a major role in protein loss in gastrocnemius muscle, up to 20% weight loss, but that other factors such as depression in protein synthesis may play a more important role at higher weight loss.

Hypericin is a potent agent in the photodynamic therapy of cancers. To better understand its tumoritropic behaviour, we evaluated the major determinants of the accumulation and dispersion of hypericin in subcutaneously growing mouse tumours. A rapid exponential decay in tumour accumulation of hypericin as a function of tumour weight was observed for each of the six tumour models investigated, and a similar relationship was found between tumour blood flow and tumour weight. Moreover, there was a close correlation between the higher hypericin uptake in RIF-1 tumours compared to R1 tumours and tumour vessel permeability. To define the role of lipoproteins in the transport of hypericin through the interstitial space, we performed a visual and quantitative analysis of the colocalization of hypericin and DiOC18-labelled lipoproteins in microscopic fluorescent overlay images. A coupled dynamic behaviour was found early after injection (normalised fluorescence intensity differences were on the whole less than 10%), while a shifted pattern in localisation of hypericin and DiOC18 was seen after 24 h, suggesting that during its migration through the tumour mass, hypericin is released from the lipoprotein complex. In conclusion, we were able to show that the tumour accumulation of hypericin is critically determined by a combination of biological (blood flow, vessel permeability) and physicochemical elements (affinity for interstitial constituents).

The potential for inhibitors of nuclear factor-kappaB (NF-kappaB) activation to act as inhibitors of muscle protein degradation in cancer cachexia has been evaluated both in vitro and in vivo. Activation of NF-kappaB is important in the induction of proteasome expression and protein degradation by the tumour factor, proteolysis-inducing factor (PIF), since the cell permeable NF-kappaB inhibitor SN50 (18 microM) attenuated the expression of 20S proteasome alpha-subunits, two subunits of the 19S regulator MSS1 and p42, and the ubiquitin-conjugating enzyme, E2(14k), as well as the decrease in myosin expression in murine myotubes. To assess the potential therapeutic benefit of NF-kappaB inhibitors on muscle atrophy in cancer cachexia, two potential inhibitors were employed; curcumin (50 microM) and resveratrol (30 microM). Both agents completely attenuated total protein degradation in murine myotubes at all concentrations of PIF, and attenuated the PIF-induced increase in expression of the ubiquitin-proteasome proteolytic pathway, as determined by the 'chymotrypsin-like' enzyme activity, proteasome subunits and E2(14k). However, curcumin (150 and 300 mg kg(-1)) was ineffective in preventing weight loss and muscle protein degradation in mice bearing the MAC16 tumour, whereas resveratrol (1 mg kg(-1)) significantly attenuated weight loss and protein degradation in skeletal muscle, and produced a significant reduction in NF-kappaB DNA-binding activity. The inactivity of curcumin was probably due to a low bioavailability. These results suggest that agents which inhibit nuclear translocation of NF-kappaB may prove useful for the treatment of muscle wasting in cancer cachexia.

This study investigated long-term microenvironmental responses (oxygenation, perfusion, metabolic status, proliferation, vascular endothelial growth factor (VEGF) expression and vascularisation) to chronic hypoxia in experimental tumours. Experiments were performed using s.c.-implanted DS-sarcomas in rats. In order to induce more pronounced tumour hypoxia, one group of animals was housed in a hypoxic atmosphere (8% O(2)) for the whole period of tumour growth (chronic hypoxia). A second group was acutely exposed to inspiratory hypoxia for only 20 min prior to the measurements (acute hypoxia), whereas animals housed under normal atmospheric conditions served as controls. Acute hypoxia reduced the median oxygen partial pressure (pO(2)) dramatically (1 vs 10 mmHg in controls), whereas in chronically hypoxic tumours the pO(2) was significantly improved (median pO(2)=4 mmHg), however not reaching the control level. These findings reflect the changes in tumour perfusion where acutely hypoxic tumours show a dramatic reduction of perfused tumour vessels (maybe the result of a simultaneous reduction in arterial blood pressure). In animals under chronic inspiratory hypoxia, the number of perfused vessels increased (compared to acute hypoxia), although the perfusion pattern found in control tumours was not reached. In the chronically hypoxic animals, tumour cell proliferation and tumour growth were significantly reduced, whereas no differences in VEGF expression and vascular density between these groups were observed. These results suggest that long-term adaptation of tumours to chronic hypoxia in vivo, while not affecting vascularity, does influence the functional status of the microvessels in favour of a more homogeneous perfusion.

Clinical photodynamic therapy (PDT) schedules are based on the assumption that optimum drug-light intervals are times at which there is a maximum differential between photosensitiser retention in the tumour and surrounding normal tissue. However, vascular-mediated effects contribute to tumour destruction by PDT; therefore, plasma sensitiser levels and endothelial cell drug exposure could also be important determinants of PDT response. The purpose of this study was to investigate the influence of tumour, tissue and plasma concentrations of the photosensitiser Foscan (meta-tetrahydroxyphenylchlorin, mTHPC) on PDT response. Groups of BalbC nude mice, bearing human mesothelioma xenografts (H-MESO1) were injected (i.v.) with a single dose of (14)C-labelled mTHPC, or with two doses, separated by 72 h. Drug levels in plasma, tumour and normal tissues were measured at 5 min to 120 h after drug administration. The PDT tumour and skin responses were evaluated by illuminating separate groups mice at intervals of 5 min to 120 h after injection of Foscan (nonlabelled). Drug levels in both tumour and skin increased during the first 24 h after a single injection, and remained almost constant for at least 120 h. The second injection produced a further, rapid increase in mTHPC levels in tumours and skin, with steady state being maintained from 20 min to 120 h. By contrast, PDT response of both tumours and skin were maximal for illumination at 1-3 h after drug, with very little response when illumination was given 48-120 h after drug. There was no significant correlation between tumour or skin drug level and PDT response. There was, however, a significant correlation between plasma drug levels and tumour or skin response, excluding an initial distribution time of 20 min. These studies demonstrate a pronounced disassociation between tumour drug levels and optimum drug-light intervals for PDT response with Foscan. We suggest that the PDT effect, in both tumours and normal tissues, is largely mediated via vascular damage and that the selectivity of PDT is not based on differential tumour drug uptake.

One of the critical steps that governs the inhibitory effect of antisense RNA on target gene expression is the association of the antisense RNA with the target RNA molecules. However, until now, no systematic method has been available to select the suitable parts of a gene as antisense targets. In this study, we utilised U1 small nuclear RNA (snRNA) that binds physiologically to the 5' splice site (5'ss) of pre-mRNA, to develop a novel vector system that permits imposed binding of antisense RNA to its target. The 5' free end of U1snRNA was replaced with the antisense sequence against the K-ras gene to generate a hyperstable U1snRNA, whose binding stability to 5'ss of the K-ras transcript is ten-fold higher than that of wild-type U1snRNA. The efficacy of such hyperstable U1snRNA was examined by transducing the expression plasmids into human pancreatic cancer cell lines. This revealed that two of the hyperstable U1snRNAs induced cell death after gene transduction, and significantly reduced the number of G418-resistant colonies to less than 10% of the controls. Furthermore, hyperstable U1snRNA suppressed intraperitoneal dissemination of pancreatic cancer cells in vivo. Hyperstable U1snRNA might be a novel approach to express effective antisense RNA in target cells., (Copyright 2002 Cancer Research UK)

The use of synthetic lipophilic molecules derived from 5-aminolevulinic acid (ALA) is currently under investigation to enhance cellular ALA penetration. In this work we studied the effect of systemic administration to mice of the hexyl ester of ALA (He-ALA) on porphyrin tissue synthesis as compared to ALA. In most normal tissues as well as in tumour, He-ALA induced less porphyrin synthesis than ALA after its systemic administration either intravenous or intraperitoneal, although explant organ cultures exposed to either ALA or He-ALA revealed equally active esterases. The only tissue that accumulated higher porphyrin levels from He-ALA (seven times more than ALA) was the brain, and this correlated well with a rapid increase in ALA/He-ALA content in brain after administration of He-ALA. This may be ascribed to a differential permeability to lipophilic substances controlled by the blood-brain barrier, a feature which could be further exploited to treat brain tumours.

Angiogenesis is a prerequisite for tumour progression and is highly regulated by growth factors and cytokines a number of which also stimulate the production of nitric oxide. Asymmetric dimethylarginine is an endogenous inhibitor of nitric oxide synthesis. Asymmetric dimethylarginine is metabolised by dimethylarginine dimethylaminohydrolase. To study the effect of dimethylarginine dimethylaminohydrolase on tumour growth and vascular development, the rat C6 glioma cell line was manipulated to overexpress the rat gene for dimethylarginine dimethylaminohydrolase I. Enhanced expression of dimethylarginine dimethylaminohydrolase I increased nitric oxide synthesis (as indicated by a two-fold increase in the production of cGMP), expression and secretion of vascular endothelial cell growth factor, and induced angiogenesis in vitro. Tumours derived from these cells grew more rapidly in vivo than cells with normal dimethylarginine dimethylaminohydrolase I expression. Immunohistochemical and magnetic resonance imaging measurements were consistent with increased tumour vascular development. Furthermore, dimethylarginine dimethylaminohydrolase activity was detected in a series of human tumours. This data demonstrates that dimethylarginine dimethylaminohydrolase plays a pivotal role in tumour growth and the development of the tumour vasculature by regulating the concentration of nitric oxide and altering vascular endothelial cell growth factor production.

Exogenous administration of 5-aminolevulinic acid (ALA) is becoming widely used to enhance the endogenous synthesis of Protoporphyrin IX (PpIX) in photodynamic therapy. We analysed porphyrin formation in chemically induced squamous papillomas, after topical application of ALA and ALA hexyl ester (He-ALA) administered in different formulations, as well as the pattern of distribution in the internal organs, and the synthesis of porphyrins in distant tumoural and normal skins. A lotion formulation containing DMSO and ethanol was the best vehicle for topical ALA delivery to papillomas, whereas cream was the most efficient formulation for He-ALA application. Similar porphyrin concentration can be accumulated in the skin tumours employing either ALA or He-ALA delivered in their optimal formulations. The use of cream as a vehicle of both ALA and He-ALA, induces highest porphyrin tumour/normal skin ratios. The main advantage of using He-ALA is that porphyrins synthesized from the ester are more confined to the site of application, thus inducing low porphyrin levels in normal skin, liver, blood and spleen, as well as in papillomas distant from the point of application, independently on the vehicle employed, so reducing potential side effects of photodynamic therapy., ((c) 2001 Cancer Research Campaign)

In the endeavour to develop a model for studying gene therapy of cancers associated with human papillomaviruses (HPVs), mouse cells were transformed with the HPV type 16 (HPV16) and activated H-ras oncogenes. This was done by cotransfection of plasmid p16HHMo, carrying the HPV16 E6/E7 oncogenes, and plasmid pEJ6.6, carrying the gene coding for human H-ras oncoprotein activated by the G12V mutation, into secondary C57BL/6 mouse kidney cells. An oncogenic cell line, designated MK16/1/IIIABC, was derived. The epithelial origin of the cells was confirmed by their expression of cytokeratins. No MHC class I and class II molecules were detected on the surface of MK16/1/IIIABC cells. Spontaneous metastases were observed in lymphatic nodes and lungs after prolonged growth of MK16/1/IIIABC-induced subcutaneous tumours. Lethally irradiated MK16/1/IIIABC cells induced protection against challenge with 10(5) homologous cells, but not against a higher cell dose (5 x 10(5)). Plasmids p16HHMo and pEJ6.6 were also used for preventive immunization of mice. In comparison with a control group injected with pBR322, they exhibited moderate protection, in terms of prolonged survival, against MK16/1/IIIABC challenge (P < 0.03). These data suggest that MK16/1/IIIABC cells may serve as a model for studying immune reactions against HPV16-associated human tumours.

Perfusion and oxygenation of experimental tumours were studied during angiotensin II (AT II) administration whereby the rate of the continuous AT II infusion was chosen to increase the mean arterial blood pressure (MABP) by 50-70 mmHg. In subcutaneous DS-sarcomas the red blood cell (RBC) flux was assessed using the laser Doppler technique and the mean tumour oxygen partial pressure (pO2) was measured polarographically using O2-sensitive catheter and needle electrodes. Changes in RBC flux with increasing MABP depended mainly on tumour size. In small tumours, RBC flux decreased with rising MABP whereas in larger tumours RBC flux increased parallel to the MABP. As a result of these volume-dependent effects on tumour blood flow, the impact of AT II on tumour pO2 was also mainly tumour volume-related. In small tumours oxygenation decreased with increasing MABP during AT II infusion, whereas in large tumours a positive relationship between blood pressure and O2 status was found. This disparate behaviour might be the result of the co-existence of two functionally distinct populations of tumour vessels. In small tumours, perfusion decreases presumably due to vasoconstriction of pre-existing host vessels feeding the tumour. In larger malignancies, newly formed tumour vessels predominate and seem not to have this vasoresponsive capability (lack of smooth muscle cells and/or AT receptors), resulting in an improvement of perfusion which is not tumour-related per se, but is due to the increased perfusion pressure.

Several possible mechanisms for the synergistic anti-tumour effects between tumour necrosis factor alpha (TNF-alpha) and melphalan after isolated limb perfusion (ILP) have been presented. We found a significant sixfold increase in melphalan tumour tissue concentration after ILP when TNF-alpha was added to the perfusate, which provides a straightforward explanation for the observed synergism between melphalan and TNF-alpha in ILP.

We previously reported that the arteriolar input in window chamber tumours is limited in number and is constrained to enter the tumour from one surface, and that the pO2 of tumour arterioles is lower than in comparable arterioles of normal tissues. On average, the vascular pO2 in vessels of the upper surface of these tumours is lower than the pO2 of vessels on the fascial side, suggesting that there may be steep vascular longitudinal gradients (defined as the decline in vascular pO2 along the afferent path of blood flow) that contribute to vascular hypoxia on the upper surface of the tumours. However, we have not previously measured tissue pO2 on both surfaces of these chambers in the same tumour. In this report, we investigated the hypothesis that the anatomical constraint of arteriolar supply from one side of the tumour results in longitudinal gradients in pO2 sufficient in magnitude to create vascular hypoxia in tumours grown in dorsal flap window chambers. Fischer-344 rats had dorsal flap window chambers implanted in the skin fold with simultaneous transplantation of the R3230AC tumour. Tumours were studied at 9-11 days after transplantation, at a diameter of 3-4 mm; the tissue thickness was 200 microm. For magnetic resonance microscopic imaging, gadolinium DTPA bovine serum albumin (BSA-DTPA-Gd) complex was injected i.v., followed by fixation in 10% formalin and removal from the animal. The sample was imaged at 9.4 T, yielding voxel sizes of 40 microm. Intravital microscopy was used to visualize the position and number of arterioles entering window chamber tumour preparations. Phosphorescence life time imaging (PLI) was used to measure vascular pO2. Blue and green light excitations of the upper and lower surfaces of window chambers were made (penetration depth of light approximately 50 vs >200 microm respectively). Arteriolar input into window chamber tumours was limited to 1 or 2 vessels, and appeared to be constrained to the fascial surface upon which the tumour grows. PLI of the tumour surface indicated greater hypoxia with blue compared with green light excitation (P < 0.03 for 10th and 25th percentiles and for per cent pixels < 10 mmHg). In contrast, illumination of the fascial surface with blue light indicated less hypoxia compared with illumination of the tumour surface (P < 0.05 for 10th and 25th percentiles and for per cent pixels < 10 mmHg). There was no significant difference in pO2 distributions for blue and green light excitation from the fascial surface nor for green light excitation when viewed from either surface. The PLI data demonstrates that the upper surface of the tumour is more hypoxic because blue light excitation yields lower pO2 values than green light excitation. This is further verified in the subset of chambers in which blue light excitation of the fascial surface showed higher pO2 distributions compared with the tumour surface. These results suggest that there are steep longitudinal gradients in vascular pO2 in this tumour model that are created by the limited number and orientation of the arterioles. This contributes to tumour hypoxia. Arteriolar supply is often limited in other tumours as well, suggesting that this may represent another cause for tumour hypoxia. This report is the first direct demonstration that longitudinal oxygen gradients actually lead to hypoxia in tumours.

Quantification of metabolite or drug concentrations in living tissues requires determination of intra- and extracellular volumes. This study demonstrates how this can be achieved non-invasively by 31P magnetic resonance spectroscopy (MRS) employing dimethyl methylphosphonate (DMMP) as a marker of total water space, 3-aminopropylphosphonate (3-APP) as a marker of extracellular space and P and 3-APP as markers of intracellular pH (pH) and extracellular pH (pHe) respectively. The MRS measurements of the tumour volumes were validated by classic radiolabelling methods using 3H2O and [14C]inulin as markers of total and extracellular space respectively. The extracellular volume fraction measured by radiolabelling of RIF-1 tumours was 23 +/- 0.83% (mean +/- s.e.m. n = 9), not significantly different (P > 0.1) from that found by MRS (27 +/- 2.9%, n = 9, London, and 35 +/- 6.7, n = 14, Baltimore). In untreated RIF-1 tumours, pH was about 0.2 units higher than pHe (P < 0.01). 5-Fluorouracil (5FU) treatment (165 mg kg(-1)) caused no significant changes in either pHe or per cent extracellular volume. However significant increases in pH, 48 h after treatment (P < 0.01) correlated with decreased tumour size and improved bioenergetic status [NTP/inorganic phosphate (Pi) ratio]. This study shows the feasibility of an MR method (verified by a 'gold standard') for studying the effects of drug treatment on intra- and extracellular spaces and pH in solid tumours in vivo.

Rodent tumour models have been the 'workhorse' for tumour oxygenation research and for investigating radiobiological hypoxic fraction. Because of the intertumour heterogeneity of blood flow and related parameters, most studies have pooled information derived from several different tumours to establish the statistical significance of specific measurements. But it is the oxygenation status of and its modulation in individual tumours that has important prognostic significance. In that regard, the bioreducible hypoxic marker technique was tested for its potential to quantify oxygenation changes within individual tumours. Beta-D-iodinated azomycin galactoside (IAZG) and beta-D-iodinated azomycin xylopyranoside (IAZXP) were each radiolabelled with Iodine-125 and iodine-131 for measurements of animal tumour oxygenation. The tumour-blood (T/B) ratio of marker radioactivity in mice after the renal excretion of unbound marker (at 3 h and longer times) had been shown to be proportional to radiobiological hypoxic fraction. When markers labelled with both radioisotopes were administered simultaneously to EMT-6 tumour-bearing scid mice, T/B ratios were found to vary by up to 300% between different tumours, with an average intratumour variation of only approximately 4%. When the markers were administered 2.5-3.0 h apart, changes in T/B ratios of 8-25% were observed in 10 out of 28 (36%) tumours. Changes to both higher and lower hypoxic fraction were observed, suggestive of acute or cycling hypoxia. When 0.8 mg g(-1) nicotinamide plus carbogen was administered to increase tumour oxygenation, reductions in T/B ratios (mean deltaT/B approximately 38%) were observed in all tumours. Similar results were obtained with Dunning rat prostate carcinomas growing in Fischer x Copenhagen rats whose T/B ratios of IAZG and radiobiological hypoxic fractions are significantly lower. These studies suggest that fluctuating hypoxia can account for at least 25% of the total hypoxic fraction in some tumours and that correlations between bioreducible marker avidity and related tumour properties will be optimal when the independent assays are performed over the same time period. This dual hypoxic marker technique should prove useful for investigating both spontaneous and induced oxygenation changes within individual rodent tumours.

The effects of combretastatin A4 prodrug on perfusion and the levels of 31P metabolites in an implanted murine tumour were investigated for 3 h after drug treatment using nuclear magnetic resonance imaging (MRI) and spectroscopy (MRS). The area of regions of low signal intensity in spin-echo images of tumours increased slightly after treatment with the drug. These regions of low signal intensity corresponded to necrosis seen in histological sections, whereas the expanding regions surrounding them corresponded to haemorrhage. Tumour perfusion was assessed before and 160 min after drug treatment using dynamic MRI measurements of gadolinium diethylenetriaminepentaacetate (GdDTPA) uptake and washout. Perfusion decreased significantly in central regions of the tumour after treatment. This was attributed to disruption of the vasculature and was consistent with the haemorrhage seen in histological sections. The mean apparent diffusion coefficient of water within the tumour did not change, indicating that there was no expansion of necrotic regions during the 3 h after drug treatment. Localized 31P-MRS showed that there was decline in cellular energy status in the tumour after treatment with the drug. The concentrations of nucleoside triphosphates within the tumour fell, the inorganic phosphate concentration increased and there was a significant decrease in tumour pH for 80 min after drug treatment. The rapid, selective and extensive damage caused to these tumours by combretastatin A4 prodrug has highlighted the potential of the agent as a novel cancer chemotherapeutic agent. We have shown that the response of tumours to treatment with the drug may be monitored non-invasively using MRI and MRS experiments that are appropriate for use in a clinical setting.

The objective of this study was first to determine whether three slowly growing early-generation murine transplantable tumours, the T40 fibrosarcoma, T115 mammary carcinoma and T237 lung carcinoma, exhibit patterns of energetics and blood flow during growth that are different from those of the faster growing RIF-1 fibrosarcoma. Serial measurements were made with 31P-magnetic resonance spectroscopy (MRS), relating to nutritive blood flow and 2H-magnetic resonance imaging (MRI), which is sensitive to both nutritive and large-vessel (non-nutritive) flow. All four tumour lines showed a decrease in betaNTP/Pi and pH with growth; however, each line showed a different pattern of blood flow that did not correlate with the decrease in energetics. Qualitative histological analysis strongly correlated with the 2H-MRI. Second, their response to 5 mg kg(-1) hydralazine i.v. was monitored by 31P-MRS. A marked decrease in betaNTP/Pi and pH was observed in both the RIF-1 fibrosarcoma and the third-generation T115 mammary carcinoma after hydralazine challenge. In contrast, the fourth generation T40 fibrosarcoma and T237 lung carcinoma showed no change in 31P-MRS parameters. However, a fifth-generation T237 cohort, which grew approximately three times faster than fourth-generation T237 cohorts, exhibited a significant deterioration in betaNTP/Pi and pH in response to hydralazine. These data are consistent with a decoupling between large-vessel and nutritive blood flow and indicate that early-generation transplants that have a slow growth rate and vascular tone are more appropriate models of human tumour vasculature than more rapidly growing, repeatedly transplanted tumours.

Solid tumours develop an acidic extracellular environment with high concentration of lactic acid, and lactic acid produced by glycolysis has been assumed to be the major cause of tumour acidity. Experiments using lactate dehydrogenase (LDH)-deficient ras-transfected Chinese hamster ovarian cells have been undertaken to address directly the hypothesis that lactic acid production is responsible for tumour acidification. The variant cells produce negligible quantities of lactic acid and consume minimal amounts of glucose compared with parental cells. Lactate-producing parental cells acidified lightly-buffered medium but variant cells did not. Tumours derived from parental and variant cells implanted into nude mice were found to have mean values of extracellular pH (pHe) of 7.03 +/- 0.03 and 7.03 +/- 0.05, respectively, both of which were significantly lower than that of normal muscle (pHe = 7.43 +/- 0.03; P < 0.001). Lactic acid concentration in variant tumours (450 +/- 90 microg g(-1) wet weight) was much lower than that in parental tumours (1880 +/- 140 microg/g(-1)) and similar to that in serum (400 +/- 35 microg/g(-1)). These data show discordance between mean levels of pHe and lactate content in tumours; the results support those of Newell et al (1993) and suggest that the production of lactic acid via glycolysis causes acidification of culture medium, but is not the only mechanism, and is probably not the major mechanism responsible for the development of an acidic environment within solid tumours.

Nitro-L-arginine inhibits the production of nitric oxide and can thereby cause vasoconstriction in vivo. One consequence of this is that nitro-L-arginine can increase hypoxia in a range of transplantable and spontaneous murine solid tumours. Bioreductive drugs such as RB6145 are more active under hypoxic conditions, and the combination of RB6145 with nitro-L-arginine in vivo shows greater anti-tumour activity than either agent individually. In mice given nitro-L-arginine at 10 mg kg(-1) i.p. up to 1 h before or after 300 mg kg(-1) i.p. RB6145, survival of KHT tumour cells was reduced by 3-4 logs when assessed by clonogenic assay 24 h after treatment. RB6145 or nitro-L-arginine alone caused no more than 20% cell kill. Similar effects were found in SCCVII tumours. The tumour response to the drug combination was tumour size dependent, with increased tumour cell sensitivity occurring when the tumour volume at the time of treatment was increased. Further, the response of KHT tumours to the combination of RB6145 and nitro-L-arginine was also dependent on the time interval between treatment and on when tumours were excised for determination of survival in vitro. The relative surviving fraction was about 0.3 for intervals less than 4 h but was reduced to 0.01 at 12 h and 0.001 at 24 h. These results were supported by histological examination of tumours, when necrosis at 2 h after treatment was less than 5% but increased to greater than 90% at 24 h. RB6145-induced normal tissue damage, as measured by CFU-A survival, was not altered by combining with nitro-L-arginine. Hence, this drug combination may provide therapeutic benefit. It is likely that the substantial anti-tumour effects are due to enhancement of bioreductive toxicity through increased tumour hypoxia, although additional mechanism(s) may also contribute to the overall response.

The purpose of this study was to determine whether power Doppler ultrasound techniques could be used to direct biopsies into tumour regions with relatively low red blood cell flux, and therefore preferentially sample regions that were relatively hypoxic. Subcutaneous 9L glioma rat tumours were biopsied using power Doppler ultrasound guidance. Immunohistochemical detection of the 2-nitroimidazole EF5 was performed to determine the presence and level of hypoxia in the biopsy samples. Comparisons between the power Doppler-determined red blood cell flux and EF5 binding were made. In seven out of eight tumours studied, power Doppler ultrasound allowed differentiation of a relatively hypoxic region from a relatively oxic region by localizing relatively low vs high red blood cell flux areas respectively. In one of these seven tumours, RBC flux was high in both biopsied sites and hypoxia was not present in either. In two of these seven tumours, hypoxia was present in each biopsy and both of the red blood cell flux measurements were low. In the eighth tumour, both the EF5 binding and the red blood cell flux measurements were low. In this tumour, low EF5 binding was due to the dominance of necrotic cells, which will not reduce or bind EF5 in the biopsy specimen. Using EF5-binding techniques, we have confirmed that regions of relatively low red blood cell flux are more hypoxic than those with relatively high red blood cell flux. Counterstaining specimens with haematoxylin and eosin allows differentiation of low EF5-binding regions due to oxia vs necrosis. These methods have clinical implications for the expanded use of power Doppler ultrasound as a means to direct tissue sampling when it is important to identify the presence of hypoxia.

The effect of nitric oxide-dependent vasodilators on vascular resistance of tumours and normal tissue was determined with the aim of modifying tumour blood flow for therapeutic benefit. Isolated preparations of the rat P22 tumour and normal rat hindlimb were perfused ex vivo. The effects on tissue vascular resistance of administration of sodium nitroprusside (SNP) and the diazeniumdiolate (or NONO-ate) NOC-7, vasodilators which act via direct release of nitric oxide (NO), were compared with the effects of acetylcholine (ACh), a vasodilator which acts primarily via receptor stimulation of endothelial cells to release NO in the form of endothelium-derived relaxing factor (EDRF). SNP and NOC-7 effectively dilated tumour blood vessels after preconstriction with phenylephrine (PE) or potassium chloride (KCl) as indicated by a decrease in vascular resistance. SNP also effectively dilated normal rat hindlimb vessels after PE/KCl constriction. Vasodilatation in the tumour preparations was accompanied by a significant rise in nitrite levels measured in the tumour effluent. ACh induced a significant vasodilation in the normal hindlimb but an anomalous vasoconstriction in the tumour. This result suggests that tumours, unlike normal tissues are incapable of releasing NO (EDRF) in response to ACh. Capacity for EDRF production may represent a difference between tumour and normal tissue blood vessels, which could be exploited for selective pharmacological manipulation of tumour blood flow.

We investigated the combined effect of radiation and clinically relevant doses of emitefur (BOF-A2), a newly developed anti-cancer agent consisting of a masked form of 5-fluorouracil (5-FU) and a potent inhibitor of 5-FU degradation, in two types of murine tumours. In preliminary pharmacokinetic studies, the area under the curve for 5-FU in plasma, after administration of 12.5 mg kg-1 and 25 mg kg-1 emitefur in mice, appeared to be similar to that obtained on the first day and that on the seventh day, respectively, after starting administration of 400-600 mg day-1 in humans. These doses (12.5 and 25 mg kg-1) of emitefur were evaluated either alone or in combination with single (15 Gy), five-fraction (4 Gy each) or ten-fraction (2.8 Gy each) irradiation using a tumour growth delay assay for SCCVII tumours and in combination with four-fraction (5 Gy each) irradiation using an in vivo-in vitro assay for EMT6 tumours. The anti-tumour and radiation-enhancing effects of 12.5 mg kg-1 emitefur were not significant in any except the ten-fraction experiment. On the other hand, multiple doses of 25 mg kg-1 emitefur given either alone or in combination with radiation produced marked effects. The mean tumour growth delay time (the time to double in volume for treated tumours minus that for untreated tumours) was 8.1 days for five administrations of 25 mg kg-1 emitefur. 10.4 days for five fractions of 4 Gy and 22.1 days for five treatments with the combination of the two. Thus, the increase in growth delay afforded by this combination was at least additive. The effect of four fractions of 5 Gy with 25 mg kg-1 emitefur in EMT6 tumours was lower than that of four fractions of 7.5 Gy, but the effect of five fractions of 4 Gy with this dose of emitefur in SCCVII tumours was similar to the effect of five fractions of 6 Gy, and the effect of ten fractions of 2.8 Gy with 25 mg kg-1 emitefur was much higher than that of ten fractions of 4.2 Gy. In conclusion, emitefur given either alone or in combination with radiation appears to have a significant anti-tumour effect even at clinically relevant dose levels, although a threshold dose exists between 12.5 and 25 mg kg-1. Further clinical studies of this compound are warranted.

2,3,5-Trimethyl-6-(3-pyridylmethyl)1,4-benzoquinone (CV-6504) is an effective inhibitor of the growth of established murine adenocarcinomas (MACs) and is shortly to enter clinical investigation. When administered to mice bearing the MAC16 tumour, CV-6504 rapidly disappeared from the plasma and tissues and there was an accumulation of the sulphate and glucuronide metabolites. After 24 h, the concentration of free CV-6504 in the tumour (3.3 microM) was higher than that in the liver (0.24 microM) and equal to the IC50 value for the inhibition of the growth of MAC16 cells in vitro (3 microM). The concentration of glucuronide and sulphate metabolites in both tumour and liver decreased with time. Both the MAC16 tumour and the liver possessed similar beta-glucuronidase activity, which could account for the accumulation of free CV-6504. Although the sulphate and glucuronide conjugates of CV-6504 were ineffective inhibitors of the growth of MAC13 cells in vitro at concentrations up to 100 microM, in vivo at a concentration of 50 mg kg-1 day-1 the conjugates produced a similar anti-tumour effect to CV-6504 at a concentration of 5 mg kg-1 day-1. The MAC13 tumour possessed both beta-glucuronidase and sulphatase activity capable of converting the sulphate and glucuronide conjugates to free CV-6504. Using MAC13 cells ex vivo, CV-6504 inhibited conversion of arachidonic acid to 5-, 12- and 15-hydroxyeicosatetraenoic acids (HETE). The percentage reduction in formation of 12- and 15-HETE exceeded that of 5-HETE. Inhibition of HETE formation may be responsible for the anti-tumour activity of CV-6504.

Novel imidazoacridinone derivatives, C1310 and C1311, have been evaluated for their potential to inhibit tumour cell growth in vitro and in vivo. A cell line panel, including seven human and murine colon carcinoma cell lines and three in vivo models, was used. The compounds were found to be potent inhibitors of tumour cell growth with IC50 values ranging between 10 nM and 2 microM in human colon cancer cell lines. Statistically significant tumour growth delay (P < 0.01) was observed after a single intraperitoneal (i.p.) dose of C1311 (100 mg kg-1 body weight) in MAC15A, MAC29 murine and HT29 human adenocarcinomas of the colon. Rapid accumulation of fluorescence of both C1310 and C1311 was seen in the nuclei of HT29 human colon tumour cells in culture. C1311 was also found to bind into calf thymus DNA as shown by spectrophotometric titration and thermal denaturation and to cause early inhibition of thymidine incorporation in HT29 cells in vitro. The results of this study suggest that C1311 should be considered as a candidate for clinical development.

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

A rapid, functional and quantitative diagnostic method for the estimation of the P-glycoprotein (P-gp)-dependent multidrug resistance is required in the clinical treatment of human tumours, as chemotherapy protocols and resistance-reversing agents could be applied accordingly. In the present work, by using a calcein accumulation method in combination with immunorecognition and drug-resistance studies, a new method is described for the quantitative estimation of the expression and function of the multidrug transporter. MDR1-transfected and drug-selected tumour cell lines with various levels of drug resistance were examined. The expression of P-gp and its cell-surface appearance were assessed by quantitative immunoblotting and by immunofluorescence cytometry. The transport function of the P-gp was assessed by measuring the extrusion of calcein acetoxymethyl ester (AM) with fluorometry and flow cytometry, while in parallel experiments drug resistance was directly examined in cell survival assays. The MDR1 activity factor (MAF), calculated from the calcein AM extrusion assay, is demonstrated to provide a reliable quantitative measure for MDR1 specific activity, reflecting cellular drug resistance. This relatively simple and rapid new functional P-gp assay surpasses the formerly used techniques in both sensitivity and reproducibility.

Hexadecafluorinated zinc phthalocyanine (ZnPcF16), an analogue of zinc phthalocyanine (ZnPc) in which all hydrogen atoms have been substituted by fluorine, was prepared as a single isomeric product via the condensation of tetrafluorophthalonitrile with zinc acetate. Fluorination renders the ZnPc soluble in most common solvents. The photodynamic properties and pharmacokinetics of the ZnPcF16 were evaluated in EMT-6 tumour-bearing Balb/c mice using 65Zn-radiolabelled analogues. Both dyes, administered i.v. at 1 mumol kg-1 as Cremophor emulsions, revealed good tumour uptake [approximately 8-9 per cent of the injected dose per g tissue (%IDg-1)] at 24 h post injection (p.i.), with the fluorinated dye reaching higher concentrations (approximately 11%IDg-1) at 48 h p.i. and subsequently higher tumour-blood ratios due to rapid blood clearance. ZnPcF16 at a dose of 5 mumol kg-1 (4.3 mg kg-1) induced complete tumour regression after phototherapy (24 h p.i., 650-700 nm band, 360 J cm-2, 200 mW cm-1). At a dose of 2 mumol kg-1 and phototherapy at 24 h p.i., the tumour volume doubling time increased to 11 days vs 6 days for the control tumours. A similar tumour growth delay was observed when phototherapy was conducted at 48 h or 72 h after dye injection implying that tumour response correlates with tumour dye concentrations rather than serum concentrations. As a result of its low solubility, the administered dose of ZnPc was limited to 1 mumol kg-1 and at this drug level significant tumour response was only observed when the dye was solubilised as the pyridinium salt. Isolation of the neoplastic cells after in vivo dye administration and in vitro exposure to red light followed by a colony formation assay showed that the ZnPcF16 exhibited a 1-2 order of magnitude higher potential for direct cell killing as compared with Photofrin and about a five times lower efficiency than ZnPc. However, all three photosensitisers induced complete occlusion of tumour vasculature immediately after PDT, suggesting that tumour regression mainly resulted from vascular stasis. The ZnPcF16 offers several advantages over ZnPc for clinical applications, including improved solubility in most solvents, resulting in facilitated drug formation, favourable pharmacokinetics as well as the potential use in fluorine magnetic resonance (F-MR) imaging.

Intra- and extracellular pH (pHi and pHe) were measured simultaneously by 31P magnetic resonance spectroscopy (MRS) in CaNT tumours before and after blood flow modification. Before modification, pHi was 7.1 +/- 0.09 (n = 11) and pHe [measured with an MRS-visible extracellular marker, 3-aminopropyl phosphonate (3-APP)] was 6.7 +/- 0.05 (n = 8). Chemical shift imaging and localised MRS experiments showed that the 3-APP signal was only from the tumour, not surrounding tissue. After modification by vascular occlusion, independent of whether tumours were maintained at room temperature (22-24 degrees C) or kept warm (33-35 degrees C), there was a decrease in pHi and pHe with pHi decreasing to a greater extent. Qualitatively similar results were found using flavone acetic acid (FAA) as a blood flow modifier; only four out of nine tumours responded to FAA. Concomitant with the reduction of the pH gradient after modification was a decrease in the phosphorylation state of the adenine nucleotides measured either as ATP/Pi by MRS or [ATP]/[ADP][P(i)] in tumour extracts. These results indicate that the intracellular uptake of chemotherapeutic drugs which are dependent on the transmembrane pH gradient will not be enhanced in cells made ischaemic as a result of vascular shutdown.

To study the effect of localised secretion of chemokines on tumour growth, the genes for human (hu) interleukin 8 (IL-8), hu-MCP-1 (MCAF), hu-MIP-1 alpha (LD78), murine (mu)-MCP-1 (JE), mu-MIP-1 alpha or mu-MIP-2 were introduced, via mammalian expression vectors, into Chinese hamster ovary (CHO) cells, and the ability of transfected cells to form tumours in vivo was evaluated. The production of hu-IL-8, hu-MIP-1 alpha or mu-MIP-1 alpha by transfected clones did not influence the growth rate in vitro, but drastically suppressed tumour growth when injected subcutaneously (s.c.) into nude mice. However, clones transfected with hu-MCP-1, mu-MCP-1 or mu-MIP-2 did not show any significant difference in growth rate in vivo compared with clones transfected with vector alone. Histological examination of the site of injection of CHO clones transfected with hu-IL-8, hu-MIP-1 alpha or mu-MIP-1 alpha showed predominantly neutrophilic infiltration. These results indicate that chemokines have potent anti-tumour activity when released, even at low doses, at the tumour site, which may be mediated by recruitment and targeting of neutrophilic granulocytes to chemokine-releasing cells. Our studies highlight the potential usefulness of localised chemokine secretion in inducing potent host anti-tumour defensive responses.

Flow cytometry using the tumour perfusion probe Hoechst 33342 was employed to examine the distribution of photosensitisers in tumour cells located at different distances from the blood supply. Two tumour models, the SCCVII squamous cell carcinoma and FsaR fibrosarcoma growing in C3H/HeN mice, were used in the experiments. Among the photosensitisers tested, only BPD (benzoporphyrin derivative monoacid) exhibited uniform distribution in tumour cells irrespective of their distance from the vasculature. In this respect, 5-aminolaevulinic acid (i.e. its metabolite protoporphyrin IX), di- and tetrasulphonated aluminium phthalocyanines (A1PcS2 and AlPcS4), di- and tetrasulphonated tetraphenylporphines (TPPS2 and TPPS4), Photofrin and bacteriochlorophyll-a (i.e. its metabolite bacteriopheophytin-a) followed BPD in decreasing order in their efficacy of accumulation in tumour cells remote from the blood supply. This photosensitiser property appeared not to depend on tumour type, tumour size, route of photosensitiser administration, time after the administration, photosensitiser lipophilicity or on the presence of host cell infiltrate in the tumour. Following treatment with photodynamic therapy (PDT) in vivo, tumour cells were sorted based on their blood vessel proximity and their survival was determined by colony formation assay. The data demonstrate that the direct killing of tumour cells by Photofrin- and A1PcS2-based PDT decreases with increasing distance of the cells from the blood supply.

The proliferating cell nuclear antigen (PCNA) is required for DNA replication and DNA nucleotide excision repair. Considerable evidence points to PCNA expression being a marker of proliferation in many situations. However, while levels of PCNA are normally very low in non-cycling tissues, high levels of the protein have been observed in the normal tissues surrounding human breast and pancreatic tumours. Using two model systems we have shown that PCNA is induced in non-cycling cells by adjacent transplanted tumour cells and that this phenomenon may be mimicked by the in vivo administration of growth factors (transforming growth factor alpha and epidermal growth factor). These data suggest that tumours may elaborate factors that induce PCNA expression in nearby normal cells. PCNA induction the normal cells surrounding tumours is a direct example of the effect of tumour cells on normal surrounding tissues. This effect may prove to be a useful parameter in the analysis of tumour-host interactions.

Pentoxifylline (PTX) has been shown to increase radiation damage to tumours and to decrease late radiation-induced injury to normal tissues. This tumour radiation sensitisation results from increased oxygen supply via improved tumour perfusion. We propose that the improved perfusion results from decreased viscous resistance and/or geometric resistance. The decreased flow resistance may be accompanied by a reduction in microvascular pressure (MVP). Since MVP is approximately equal to the interstitial fluid pressure (IFP), PTX should lead to a decrease in IFP. To test this hypothesis, we measured PO2, laser Doppler flow (RBC flux) and IFP in FSaII murine tumours at two doses (PTX at 25 and 100 mg per kg body weight) which sensitise this tumour to X-irradiation. We found that 25 mg kg-1 PTX was ineffective, but 100 mg kg-1 PTX was effective in increasing the PO2 of this tumour. PTX at 100 mg kg-1 (i.p.) increased median PO2 from 5 to 7 mmHg (P < 0.05) within 2 h, and decreased the fraction of PO2 values < 5 mmHg from 65% to 45% (P < 0.05). In support of our hypothesis, we found that with this dose of PTX, RBC flux in the tumour centre increased significantly (n = 6, P < 0.05) prior to an approximately 40% decrease (n = 13, P < 0.05) in tumour interstitial fluid pressure (TIFP), without changes in mean arterial blood pressure (MABP). In conclusion, a single i.p. administration of PTX at 100 mg kg-1 can increase oxygen availability in the tumour due to ameliorate hypoxia in tumour microregions. Second, PTX can lower the elevated TIFP without lowering the MABP.

Experiments on s.c. rat tumours (DS sarcoma) were performed to determine whether chronic or acute changes in tumour perfusion necessarily lead to changes in tissue oxygenation and bioenergetic status since, as a rule, blood flow is thought to be the ultimate determinant of the tumour bioenergetic status. Based on this study, there is clear experimental evidence that growth-related or acute (following i.v. administration of tumour necrosis factor alpha) decreases in tumour blood flow are accompanied by parallel alterations in tissue oxygenation. In contrast, tumour energy status remains stable as long as flow values do not fall below 0.4-0.5 ml g-1 min-1, and provided that glucose as the main substrate can be recruited from the enlarged interstitial compartment. Perfusion rate seems to play a paramount role in determining energy status only in low-flow tumours or low-flow tissue areas.

Solid tumours are known to develop regions of extracellular acidity and survival of tumour cells in such regions depends on membrane-based mechanisms which regulate intracellular pH (pHi). We have therefore developed a method, based on dual staining of cells and flow cytometry, to study the regulation of pHi in subpopulations of tumours and spheroids. The activity of membrane-based pHi regulating transporters was studied in EMT-6 and MGH U1 cells grown in monolayer culture, spheroids, and tumours. pHi was measured with the fluorescent pH probe 2'7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, and Hoechst 33342 was used to identify cells from different regions of tumours and spheroids. In monolayer culture, incubation of cells for 18 h at pHe 6.6 led to a 1.3-1.5-fold enhancement in the activity of both the Na+/H+ exchanger and the Na(+)-dependent Cl-@HCO3- exchanger. This effect was prevented by the protein synthesis inhibitor cycloheximide. Cells from the centre of EMT-6 spheroids had increased activity of the Na+/H+ exchanger compared to cells from the periphery, when spheroids were grown in medium at pH 6.6, but not at 7.4. By contrast, in MGH U1 spheroids, cells from the centre had increased activity of the Na+/H+ antiport under both sets of conditions. There was no significant difference in the activity of the Na+/H+ exchanger in cells derived from different subpopulations of EMT-6 tumours or MGH U1 xenografts in nude mice. Although upregulation of Na+/H+ exchange occurs after exposure to acidic conditions in vitro, the microenvironmental conditions found within solid tumours do not appear to cause this effect. Our results suggest the feasibility of pharmacological inhibition of Na+/H+ exchange activity as an approach to therapy directed against nutrient-deprived tumour cells.

SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide, WIN 59075, tirapazamine) is the lead compound in a new class of bioreductive anticancer drugs, the benzotriazine di-N-oxides. It is currently undergoing Phase I clinical testing. The preferential tumour cell killing of SR 4233 is a result of its high specific toxicity to cells at low oxygen tensions. Such hypoxic cells are a common feature of solid tumours, but not normal tissues, and are resistant to cancer therapies including radiation and some anticancer drugs. The killing of these tumour cells by SR 4233, particularly when given on multiple occasions, can increase total tumour cell killing by fractionated irradiation by several orders of magnitude without increasing toxicity to surrounding normal tissues. Topics covered in this review include the rationale for developing a hypoxic cytotoxic agent, the cytotoxicity of SR 4233 as a function of oxygen concentration, the mechanism of action of the drug and its intracellular target and the in vivo evidence that the drug may be useful as an adjunct both to radiotherapy and chemotherapy. Finally, the major unanswered questions on the drug are outlined.

Acquired resistance to cisplatin (DDP) is a major clinical problem in the treatment of ovarian, testicular, and head and neck carcinomas; decreased accumulation of DDP is the most consistently observed alteration in resistant cells. It has been postulated that DDP enters the cell by passive diffusion based on the observations that DDP accumulation is proportional to the drug concentration, accumulation is not saturable, and that structural analogs of DDP do not inhibit accumulation. However, recent studies show that DDP accumulation can be specifically stimulated or inhibited by pharmacological agents and the activation of signal transduction pathways. This paper reviews the existing data on the mechanism of DDP accumulation and develops the postulate that some component of transport occurs through a gated ion channel.

The effects of progressive cachexia on protein metabolism in skeletal muscle has been investigated in mice bearing the MAC16 adenocarcinoma which produces cachexia with tumour burdens of < 1% of the host weight. Weight loss was accompanied by loss of whole body nitrogen in proportion to the overall loss of body mass. Using L-[4-3H]phenylalanine to label proteins in gastrocnemius muscle, a significant depression (60%) in protein synthesis occurred in animals with a weight loss between 15 and 30% accompanied by an increase in protein degradation, which increased with increasing weight loss between 15 and 30%. Muscle degradation in vitro could be achieved by serum from cachectic animals, which appeared to contain a proteolysis-inducing factor. These results suggest that the increased degradation of skeletal muscle seen in this model of cachexia may be due to a circulating proteolysis-inducing factor.

The extracellular pH (pHe) in solid tumours is frequently lower than the pHe in normal tissues. Cells within an acidic environment depend on mechanisms which regulate intracellular pH (pHi) for their survival, including the Na+/H+ antiport which exports protons in exchange for Na+ ions. Amiloride and its analogues DMA (5-(N,N-dimethyl)amiloride), MIBA (5-(N-methyl-N-isobutyl)amiloride) and EIPA (5-(N-ethyl-N-isopropyl)amiloride) are known to inhibit the Na+/H+ antiport and therefore decrease the cells ability to regulate pHi. All three analogues were found to be potent inhibitors of the antiport in human MGH-U1 and murine EMT-6 cells, with DMA being approximately 20, MIBA 100 and EIPA 200-fold as potent as amiloride; EIPA also gave more complete suppression of the Na+/H+ antiport. These agents were not toxic to cells when used alone; however, in combination with nigericin, an agent which acidifies cells, all three analogues were toxic to cells at pHe < 7.0, and markedly enhanced the toxicity of nigericin alone. Cell killing was greatest for nigericin used with EIPA or MIBA. None of the agents were toxic to cells at pHe 7.0 or above. When used against variant cells lacking the Na+/H+ antiport (PS-120 cells) EIPA did not enhance the cytotoxicity of nigericin alone, suggesting that the observed effect was due to inhibition of Na+/H+ exchange, rather than due to non-specific effects. The combination of EIPA and nigericin gave similar cell killing in previously dissociated and intact MGH-U1 spheroids, suggesting that the agents have good penetration of solid tissue. Preliminary experiments using EMT-6 tumours in mice suggested that EIPA and nigericin were able to enhance the toxicity of radiation in vivo, presumably through selective effects against the hypoxic (and probably acidic) subpopulation of cells that is resistant to radiation.

SR 4233 (3-amino-1,2,4-benzotriazine 1,4-dioxide) is an anti-tumour agent that has a highly selective toxicity to hypoxic cells. In this study we delineate the role of several different bioreductive enzymes in the metabolism of SR 4233 by two tumour cell lines HT 1080 (human fibrosarcoma) and SCCVII (mouse carcinoma). Enzyme kinetics demonstrates similar KM of HT 1080 and SCCVII cell sonicates and differing Vmax. Among all cofactors tested, NADPH was the most important one in reducing SR 4233 by both tumour cell sonicates. NADH was the second most important cofactor while hypoxanthine and N-methylnicotinamide were less involved in the reduction of SR 4233. Carbon monoxide inhibited the reduction by about 60% suggesting that cytochrome P-450 may play a major role in the reduction of SR 4233 under hypoxia in both SCCVII and HT 1080 cells. DT diaphorase is also involved, particularly in HT 1080 cells, in this drug reduction. The level of functional cytochrome P-450, cytochrome P-450 reductase activity and DT diaphorase activity in both cell lines were assayed. These enzyme levels were all higher in SCCVII cells than in HT 1080 cells. This result correlated the higher Vmax of SR 4233 reduction in SCCVII cells than in HT 1080 cells.

The growth rate of the MAC16 tumour in cachectic animals was significantly enhanced by the hypolipidemic agent bezafibrate, while the growth rate of a histologically similar tumour, the MAC13, which grows without an effect on host body compartments was unaffected. Growth of the MAC16 in vitro was unaffected by bezafibrate, suggesting that it was an in vivo phenomenon only. The stimulatory effect of bezafibrate correlated with the maximum plasma levels of free fatty acids (FFA) arising from the catabolism of adipose tissue. Accumulation of 14C-lipid from 1-14C-triolein administered by intragastric intubation was enhanced in heart, gastrocnemius muscle and tumour of bezafibrate treated animals, while the total lipid absorption did not differ from solvent treated controls. The increased lipid accumulation in the heart, but not the tumour correlated with an increased tissue lipoprotein lipase level. The increased tumour level may arise from an increased uptake of FFA arising from a weakening of the bonds between FFA and albumin. These results suggest that growth of certain tumours is dependent on maintaining sufficient lipid levels and that the lipid mobilising effect of the tumour may be necessary to sustain tumour growth.

The acute toxicity, pharmacokinetics and hypoxic cytotoxicity of RSU-1069 were investigated using the subcutaneous (sc) rat 9L tumour model. The pharmacokinetics were studied after i.p. injection of RSU-1069 (20 mg kg-1 or 100 mg kg-1). For both doses, the elimination of RSU-1069 followed first-order kinetics in both plasma and unclamped tumours. After 100 mg kg-1, the peak plasma concentration of RSU-1069 was 40 micrograms ml-1; the elimination t1/2 was 39.3 +/- 11.1 min. After 20 mg kg-1, the peak plasma concentration was 3 micrograms ml-1; the elimination t1/2 was 47.8 +/- 6.3 min. In unclamped tumours, the peak concentration was 50 micrograms g-1 with an elimination t1/2 of 36.1 +/- 9.6 min for the 100 mg kg-1 dose, and 4 micrograms g-1 with an elimination t1/2 of 41.9 +/- 6.1 min for the 20 mg kg-1 dose. The tumour and plasma elimination half-times were not significantly different (P greater than 0.2) for the two doses. Clamping the tumour 30 min after administration of 100 mg kg-1 of RSU-1069 decreased the tumour elimination t1/2 to 10.9 +/- 1.4 min. After releasing the clamp, RSU-1069 returned rapidly to the unclamped tumour concentration. The unclamped tumour/plasma ratio reached a maximum of 4-6, then decreased to a constant value of about 2 for both doses, indicating that RSU-1069 accumulates in these 9L tumours. RSU-1069 kills hypoxic sc 9L cells more efficiently than oxic sc 9L cells; at a surviving fraction of 0.5, the SER was 4.8. For in vitro 9L cells, the SER was approximately 50 when the comparison was between those treated in 2.1% 0(2) and those treated in less than 7.5 x 10(-3)% 0(2); it was approximately 100 when the comparison was between those treated in 21% 0(2) and those treated in less than 7.5 x 10(-3)% 0(3). Tumours treated with RSU-1069 and clamped for various times exhibited biphasic cell-kill kinetics; at 50 mg kg-1, little additional cell kill was achieved after 40 min of clamping. Our data also indicate that RSU-1069 is 300-1000 fold more efficient than misonidazole or SR2508 for killing hypoxic sc 9L tumour cells in situ.

This study examined the pathophysiological role of parathyroid hormone-related protein (PTHrP) in humoral hypercalcaemia of malignancy (HHM). Seven human tumour xenografts were analysed in nude mice; five tumours (KEsC-2, oesophageal carcinoma; FA-6, pancreatic carcinoma; SEKI, melanoma; Lu-65A and Lu-61, lung carcinomas) were associated with hypercalcaemia and two tumours (MIA PaCa-2, pancreatic carcinoma; PLC/PRF/5, hepatocellular carcinoma) with normocalcaemia. Northern blot analyses, radioimmunoassay and bioassay confirmed the synthesis of PTHrP-like peptides by all five tumours associated with hypercalcaemia, but not by the two associated with normocalcaemia. These observations indicated a very close relationship between the production of PTHrP and the development of HHM. Gel filtration studies of three tumour tissue extracts revealed at least two different molecules with both PTHrP-like immunological and biological activities. One peak eluted at a position between PTHrP (1-141) and cytochrome C and the other at a position identical to cytochrome C. These results suggest that PTHrP molecules with a molecular size equal to or greater than cytochrome C participate as causative agents of HHM. All five tumour xenografts caused hypercalcaemia when grown to a size of 1.5 g in nude mice. Under cell culture conditions, four original cell lines, KEsC-2, FA-6, SEKI and Lu-65A secreted 450.0, 45.0, 3.6 and 3.0 pmol of immunoreactive PTHrP/1.5 x 10(9) cells (approximately equivalent to 1.5 g wet weight) 24 h-1 into their respective culture media. Since a subcutaneous infusion of 100 pmol 24 h-1 of PTHrP (1-34) into nude mice was sufficient to induce significant hypercalcaemia, we speculate that PTHrP alone released from tumour cells could induce hypercalcaemia at least in the case of KEsC-2, and possibly in FA-6. With regard to other tumours associated with hypercalcaemia, further examination of PTHrP and other compounds with bone-resorbing activity in these transplantable tumours is required to obtain a better understanding of this morbidity.

The in vivo characteristics of four analogues of benzoporphyrin derivative (BPD) have been investigated. Biodistribution data obtained in DBA/2J mice with BPD-MA (monoacid ring A analogue) which had been tritiated or internally labelled with 14C showed that both labelled materials acted in an essentially identical manner during the period of study. Biodistribution and clearance studies showed that relative distribution in a variety of mouse tissues was similar for all BPD analogues. M1 tumour cells (rhabdomyosarcoma in DBA/2J mice) taken from tumours excised from animals treated 3 h earlier with BPD, and tested in vitro for photosensitivity provided evidence that significant levels of photosensitiser detected in tumour was both active and associated with tumour cells. The monoacid forms of BPD were found to be much more photodynamically active in this test than were the diacid analogues. The ability of the analogues to ablate tumours in mice by photodynamic therapy was also tested. Again, BPD-MA and BPD-MB proved to be measurably better than the diacid analogues. These findings are discussed in reference to structural and physical differences between the analogues.