Initial stages of tumor cell-induced angiogenesis: evaluation via skin window chambers in rodent models.

BACKGROUND: There is a paucity of information about events that follow immediately after tumor cells are triggered to initiate the process of angiogenesis (the formation of new blood vessels). Such information is relevant to the issue of when micrometastases vascularize and has implications for the accessibility of micrometastases to various treatments. In this study, we attempted to monitor events at the initiation of angiogenesis at the earliest possible stage of tumor growth in vivo. METHODS: Two different rodent mammary tumor cell lines, R3230Ac from the Fischer 344 rat and 4T1 from the BALB/c mouse, were stably transfected with a gene that encodes an enhanced version of green fluorescence protein (GFP). GFP-labeled R3230Ac or 4T1 cells (about 20-50 cells) were implanted into dorsal skinfold window chambers of Fischer 344 rats or BALB/c mice, respectively. Tumor angiogenesis was then monitored serially and noninvasively for up to 4 weeks. RESULTS: Clear evidence of modification of the host vasculature was observed when tumor mass reached approximately 60-80 cells, and functional new blood vessels were seen when tumor mass reached roughly 100-300 cells. Individual tumor cells exhibited a chemotaxis-like growth pattern toward the pre-existing host vasculature. When ex-flk1 (a soluble, truncated vascular endothelial cell growth factor receptor protein known to be antiangiogenic) was injected with the tumor cells, the initial angiogenic and tumor growth activities were inhibited considerably, indicating that angiogenesis inhibitors may halt tumor growth even before the onset of angiogenesis. CONCLUSION: Angiogenesis induced by tumor cells after implantation in the host begins at a very early stage, i.e., when the tumor mass contains roughly 100-300 cells. Identification of chemotactic signals that initiate tumor cell migration toward the existing vasculature may provide valuable targets for preventing tumor progression and/or metastases.

Characterization of the effect of hyperthermia on nanoparticle extravasation from tumor vasculature.

The efficacy of novel cancer therapeutics can be hampered by inefficient delivery of agents to the tumor at effective concentrations. Liposomes have been used as a method to overcome some delivery issues and, in combination with hyperthermia, have been shown to increase drug delivery to tumors. This study investigates the effects of a range of temperatures (34-42 degrees C) and hyperthermia treatment scheduling (time between hyperthermia and drug administration as well as between consecutive hyperthermia treatments) on the extravasation of nanoparticles (100-nm liposomes) from tumor microvasculature in a human tumor (SKOV-3 ovarian carcinoma) xenograft grown in athymic nude mouse window chambers. Under normothermic conditions (34 degrees C) and at 39 degrees C, nanoparticles were unable to extravasate into the tumor interstitium. From 40 to 42 degrees C, nanoparticle extravasation increased with temperature, reaching maximal extravasation at 42 degrees C. Temperatures higher than 42 degrees C led to hemorrhage and stasis in tumor vessels. Enhanced nanoparticle extravasation was observed several hours after heating, decaying back to baseline at 6 h postheating. Reheating (42 degrees C for 1 h) 8 h after an initial heating (42 degrees C for 1 h) did not result in any increased nanoparticle extravasation, indicating development of vascular thermotolerance. The results of this study have implications for the application and scheduling of hyperthermia combined with other therapeutics (e.g., liposomes, antibodies, and viral vectors) for the treatment of cancer.

Hyperthermia enables tumor-specific nanoparticle delivery: effect of particle size.

The efficacy of novel cancer therapeutics has been hampered by the ability to deliver these agents to the tumor at effective concentrations. Liposomes have been used as a method to overcome some delivery issues and, in combination with hyperthermia, have been shown to increase drug delivery to tumors. Particle size has been shown to affect the delivery of liposomes, but it is not known how hyperthermia affects size dependence. This study investigates the effect of hyperthermia (42 degrees C) on the extravasation of different sized nanoparticles (albumin; 100-, 200-, and 400-nm liposomes) from tumor microvasculature in a human tumor (SKOV-3 ovarian carcinoma) xenograft grown in mouse window chambers. In this model (at 34 degrees C), no liposomes were able to extravasate into the tumor interstitium. Hyperthermia enabled liposome extravasation of all sizes. The magnitude of hyperthermia-induced extravasation was inversely proportional to particle size. Thus, at normothermia (34 degrees C), the pore cutoff size for this model was between 7 and 100 nm (e.g., liposomes did not extravasate). At 42 degrees C, the pore cutoff size was increased to >400 nm, allowing all nanoparticles tested to be delivered to the tumor interstitium to some degree. With hyperthermia, the 100-nm liposome experienced the largest relative increase in extravasation from tumor vasculature. Hyperthermia did not enable extravasation of 100-nm liposomes from normal vasculature, potentially allowing for tumor-specific delivery. These experiments indicate that hyperthermia can enable and augment liposomal drug delivery to tumors and potentially help target liposomes specifically to tumors.

Efficacy of liposomes and hyperthermia in a human tumor xenograft model: importance of triggered drug release.

The tumor drug concentrations, drug distributions, and therapeutic efficacies achieved by three fundamentally different liposomes, nonthermosensitive liposome (NTSL), traditional thermosensitive liposome (TTSL), and low temperature sensitive liposome (LTSL); free doxorubicin (DOX); and saline in combination with hyperthermia (HT) were directly compared in a human tumor xenograft model. NTSL is a nonthermosensitive liposome in the physiological temperature range, TTSL is a traditional thermosensitive liposome that triggers in the range of approximately 42-45 degrees C and releases drug over approximately 30 min, and LTSL is a new low temperature sensitive liposome that triggers in the range of approximately 39-40 degrees C and releases drug in a matter of seconds. Because of the different attributes of the liposomes, it was possible to delineate the relative importance of liposome drug encapsulation, HT cytotoxicity, HT-drug interaction, HT-induced liposomal delivery, and HT-triggered liposomal drug release in achieving antitumor activity. Athymic nude mice bearing the FaDu human tumor xenograft were given a single i.v. dose of 5 mg/kg of DOX (free drug or liposome encapsulated), and the tumors were then heated to either 34 degrees C or 42 degrees C for 1 h at 34 degrees C. All treatment groups were similar, achieving low concentrations of DOX (0-4.5 ng/mg). At 42 degrees C, the LTSL (25.6 ng/mg) achieved the highest DOX concentration (P < 0.04), but all three liposomal formulations (7.3-25.6 ng/mg) were higher than saline or DOX (0-0.7 ng/mg; P < 0.02). LTSL + HT was also the only group that resulted in significant amounts of DNA-bound DOX (silver nitrate-extractable fraction; P < 0.02). Tumor tissue sections were visualized for DOX fluorescence to investigate the local distribution of the drug in the tumor and confirm the relative drug concentrations based on fluorescence intensity. There was relatively little fluorescence seen with treatment groups at 34 degrees C. At 42 degrees C, the LTSL showed the most DOX fluorescence (P < 0.01), and the fluorescence, although not homogeneous, was pervasive throughout the tumor sections. Therapeutic efficacy of treatments was determined from tumor growth time. At 34 degrees C, the only treatment group significantly better than the saline group (9.8 days) was the NTSL group, with a growth time of 20.9 days (P < 0.02). At 42 degrees C, all three liposomal formulations were more efficacious than DOX. LTSL + HT had the longest growth time (51.4 days) and the most number of local controls at 60 days (six of nine tumors). With HT, the DOX concentrations and fluorescence were tightly correlated with tumor growth delay, indicating that adequate (increased) drug delivery can be predictive of therapeutic effect. Overall, the LTSL + HT group showed the largest DOX concentration, the highest and most pervasive DOX fluorescence, and the most antitumor effect. Thus, HT-triggered liposomal drug release may account for the largest differential therapeutic effect and demonstrates the importance of rapid drug release from the drug carriers at the tumor site.

Fluctuations in red cell flux in tumor microvessels can lead to transient hypoxia and reoxygenation in tumor parenchyma.

Hypoxia occurs in two forms in tumors. Chronic or diffusion-limited hypoxia is relatively well characterized. In contrast, intermittent or perfusion-limited hypoxia is not well characterized, and it is not known how common it is in tumors. The purpose of this study was to determine whether spontaneous fluctuations in tumor microvessel flow rate can modify vessel oxygen tension (pO2) sufficiently to cause intermittent hypoxia (IH; tissue pO2 < 3 mmHg) in the tumor parenchyma supplied by such vessels. Microvessel red cell flux (RCF) and perivascular pO2 were measured simultaneously and continuously in dorsal flap window chambers of Fischer-344 rats with implanted R3230Ac tumors. In all vessels, RCF was unstable, with apex/nadir ratios ranging from 1.5 to 10. RCF and pO2 were temporally coordinated, and there were linear relationships between the two parameters. Vascular pO2 was less sensitive to changes in RCF in well-vascularized tumor regions compared with poorly vascularized regions. Simulations of oxygen transport in a well-vascularized region of a tumor demonstrated that two-fold variations in RCF can produce IH in 30% of the tissue in that region. In poorly vascularized regions, such fluctuations would lead to an even greater percentage of tissue involved in transient hypoxia. These results suggest that IH is a relatively common phenomenon. It could affect binding of hypoxic cytotoxins to tumor cells, in addition to being an important source of treatment resistance. Intermittent hypoxia also could contribute to tumor progression by providing repeated exposure of tumor cells to hypoxia-reoxygenation injury.

Oxygen distribution and consumption in the cat retina during normoxia and hypoxemia.

Oxygen tension (PO2) was measured with microelectrodes within the retina of anesthetized cats during normoxia and hypoxemia (i.e., systemic hypoxia), and photoreceptor oxygen consumption was determined by fitting PO2 measurements to a model of steady-state oxygen diffusion and consumption. Choroidal PO2 fell linearly during hypoxemia, about 0.64 mmHg/mmHg decrease in arterial PO2 (PaO2). The choroidal circulation provided approximately 91% of the photoreceptors' oxygen supply under dark-adapted conditions during both normoxia and hypoxemia. In light adaptation the choroid supplied all of the oxygen during normoxia, but at PaO2's less than 60 mmHg the retinal circulation supplied approximately 10% of the oxygen. In the dark-adapted retina the decrease in choroidal PO2 caused a large decrease in photoreceptor oxygen consumption, from approximately 5.1 ml O2/100 g.min during normoxia to 2.6 ml O2/100 g.min at a PaO2 of 50 mmHg. When the retina was adapted to a rod saturating background, normoxic oxygen consumption was approximately 33% of the dark-adapted value, and hypoxemia caused almost no change in oxygen consumption. This difference in metabolic effects of hypoxemia in light and dark explains why the standing potential of the eye and retinal extracellular potassium concentration were previously found to be more affected by hypoxemia in darkness. Frequency histograms of intraretinal PO2 were used to characterize the oxygenation of the vascularized inner half of the retina, where the oxygen distribution is heterogeneous and simple diffusion models cannot be used. Inner retinal PO2 during normoxia was relatively low: 18 +/- 12 mmHg (mean and SD; n = 8,328 values from 36 profiles) in dark adaptation, and significantly lower, 13 +/- 6 mmHg (n = 4,349 values from 19 profiles) in light adaptation. Even in the dark-adapted retina, 30% of the values were less than 10 mmHg. The mean PO2 in the inner (i.e., proximal) half of the retina was well regulated during hypoxemia. In dark adaptation it was significantly reduced only at PaO2's less than 45 mmHg, and it was reduced less at these PaO2's in light adaptation.

Temporal changes in PO2 of R3230AC tumors in Fischer-344 rats.

PURPOSE: The purpose of this study was to characterize the kinetics of hypoxia-reoxygenation in a murine tumor. Information on the prevalence and kinetics of this process are lacking in solid tumors, although there are data on blood flow fluctuation. MATERIALS AND METHODS: Oxygen tension (pO2) was monitored at one position in 1 cm diameter R3230Ac tumors of Fischer-344 rats, using 10-12 microm diameter recessed-tip polarographic electrodes. Data were collected continuously at a sampling frequency of 25 Hz for 30-90 min. Mean arterial blood pressure (MAP) and heart rate were also monitored. RESULTS: Temporal fluctuations in pO2 were observed in all 13 experiments. To assess the potential for hypoxia-reoxygenation, two threshold pO2 values were chosen (5 and 10 mmHg), and the number and duration of intervals that measurements resided below the thresholds was quantitated. In some experiments, the measurements did not fluctuate across the threshold values and, instead, either remained above or below them throughout the observation period. The percentage of sites that did not fluctuate across the thresholds was 38 and 61% for the 10- and 5-mmHg values, respectively. For the remaining studies, fluctuations above and below the thresholds of hypoxia ranged around 4-7 events per h. There were wide variations in the duration of hypoxic episodes, ranging from less than 1 to more than 40 min. The percentage time that measurements were below the hypoxic thresholds was also variable, ranging from 30-90%. CONCLUSIONS: These results, taken with the already published data on temporal instability in human and murine tumor blood flow, suggest that intermittent hypoxia is a common phenomenon in tumors. Future studies will focus on the underlying mechanisms that contribute to this process, because it has important implications for radiation and chemotherapy and, perhaps, gene regulation in tumors.

Variability in blood flow and pO2 in tumors in response to carbogen breathing.

PURPOSE: There is speculation that the CO2 in carbogen (95% O2, 5% CO2) can block the vasoconstrictive effects of oxygen. However, it has recently been shown that blood flow in human tumors is variable while patients breathe carbogen. Furthermore, we have shown a consistent decrease in tumor blood flow (TBF) with carbogen breathing in the rat window chamber model. Also, we have previously shown that there is no significant difference in tumor growth time after radiation with air vs. carbogen breathing. This study was designed to investigate the effects of carbogen breathing on blood flow and oxygen levels in a solid tumor. METHODS: Measurements were made in Fischer-344 rats with 8-10 mm diameter R3230Ac tumors transplanted either within the quadriceps muscle (n = 16) or subcutis (n = 14). Nontumor-bearing quadriceps muscle was studied in six other rats. After a 20-minute air-breathing baseline, rats breathed carbogen for an additional 40 minutes. Partial pressure of oxygen (pO2) was continuously monitored at one position for 60 minutes using 9-12 microm diameter oxygen microelectrodes. Blood flow was simultaneously monitored in all animals using laser Doppler flowmetry (1-2 probes/tumor). RESULTS: Blood flow changes during carbogen breathing were variable in all tissues and intratumoral heterogeneity was observed. Despite variability in blood flow, pO2 consistently increased in normal muscle but varied in both tumor sites. During carbogen breathing, the percent pO2 measurements greater than the baseline average were 99.5% +/- 0.4% (mean +/- SEM), 42.7% +/- 13.8%, and 79.8% +/- 11.0% in normal muscle, subcutaneous tumor, and muscle tumor, respectively. To show the magnitude of change, average pO2 values during air and carbogen breathing were calculated for each site. Normal muscle increased from 14.9 +/- 2.3 to 39.0 +/- 6.4 mm Hg (paired t-test; p = 0.009). Muscle tumors showed a rise from 14.6 +/- 3.2 to 34.5 +/- 8.2 mm Hg (p = 0.019). However, pO2 in subcutaneous tumors remained unchanged, with a pO2 of 7.3 +/- 2.0 mm Hg on air and 7.3 +/- 4.1 mm Hg (p = 0.995) during carbogen breathing. CONCLUSIONS: Carbogen had no consistent effect on blood flow and was ineffective at increasing tumor pO2. These results may partially explain why carbogen breathing failed to improve the efficacy of radiation in this tumor model when transplanted subcutaneously.

Tissue gradients of energy metabolites mirror oxygen tension gradients in a rat mammary carcinoma model.

PURPOSE: It has been shown that oxygen gradients exist in R3230AC tumors grown in window chambers. The fascial surface is better oxygenated than the tumor surface. The purpose of the present study was to determine whether gradients exist for energy metabolites and other end points related to oxygen transport. METHODS AND MATERIALS: Imaging bioluminescence was used to measure ATP, glucose, and lactate in cryosections of R3230AC tumors. Mean vessel density and hypoxic tissue fraction were assessed using immunohistochemistry. Tumor redox ratio was assessed by redox ratio scanning. RESULTS: Lactate content and hypoxic fraction increased, whereas ATP, glucose, redox ratio, and vessel density decreased from the fascial to the tumor surface. CONCLUSIONS: The data support a switch from aerobic to anaerobic metabolism concomitant with the PO2 gradient. The vascular hypoxia that exists in perfused vessels at the tumor surface leads to macroscopic tissue regions with restricted oxygen availability and altered metabolic status. Methods to reduce tumor hypoxia may have to take this into account if such gradients exist in human tumors. The results also have implications for hypoxia imaging, because macroscopic changes in PO2 (or related parameters) will be easier to see than PO2 gradients limited to the diffusion distance of oxygen.

Simultaneous administration of glucose and hyperoxic gas achieves greater improvement in tumor oxygenation than hyperoxic gas alone.

PURPOSE: To test the feasibility of hyperglycemic reduction of oxygen consumption combined with oxygen breathing (O(2)), to improve tumor oxygenation. METHODS AND MATERIALS: Fischer-344 rats bearing 1 cm R3230Ac flank tumors were anesthetized with Nembutal. Mean arterial pressure, heart rate, tumor blood flow ([TBF], laser Doppler flowmetry), pH, and pO(2) were measured before, during, and after glucose (1 or 4 g/kg) and/or O(2). RESULTS: Mean arterial pressure and heart rate were unaffected by treatment. Glucose at 1 g/kg yielded maximum blood glucose of 400 mg/dL, no change in TBF, reduced tumor pH (0.17 unit), and 3 mm Hg pO(2) rise. Glucose at 4 g/kg yielded maximum blood glucose of 900 mg/dL, pH drop of 0.6 unit, no pO(2) change, and reduced TBF (31%). Oxygen tension increased by 5 mm Hg with O(2). Glucose (1 g/Kg) + O(2) yielded the largest change in pO(2) (27 mm Hg); this is highly significant relative to baseline or either treatment alone. The effect was positively correlated with baseline pO(2), but 6 of 7 experiments with baseline pO(2) < 10 mm Hg rose above 10 mm Hg after combined treatment. CONCLUSION: We demonstrated the feasibility of combining hyperglycemia with O(2) to improve tumor oxygenation. However, some cell lines are not susceptible to the Crabtree effect, and the magnitude is dependent on baseline pO(2). Additional or alternative manipulations may be necessary to achieve more uniform improvement in pO(2).

Retinal oxygen tension and the electroretinogram during arterial occlusion in the cat.

PURPOSE: Retinal oxygen tension (PO2), photoreceptor oxygen consumption (QO2), the local electroretinogram (LERG), and the vitreally recorded electroretinogram (ERG) were evaluated during retinal artery occlusion in the cat. The feasibility of supplying the retina with oxygen during occlusion by ventilation with 100% O2 was evaluated. METHODS: Double-barreled oxygen microelectrodes were used to measure intraretinal PO2 and LERGs in anesthetized cats before, during, and after occlusion of a single retinal artery. Outer retinal (photoreceptor) QO2 was determined from retinal PO2 profiles. RESULTS: During air breathing, occlusion obliterated the LERG b-wave and reduced the vitreal ERG by the amount expected from the area supplied by the occluded vessel. The PO2 in the entire inner retina was zero, and photoreceptor QO2 was decreased by approximately 25%. Inspiration of 100% O2 restored the b-wave amplitude to approximately 50% of normal and increased the amount of O2 reaching the inner retina. Hyperoxia could not guarantee nonzero PO2 across the entire retina in either darkness or light, but it elevated the average PO2 in the innermost 25% of the retina to more than 20 mm Hg. The b-wave amplitude must have been affected by some factor in addition to local hypoxia, because the amplitude was not correlated with inner retinal PO2 during occlusion, and a normal PO2 did not result in a normal LERG. Effects of 2 to 2.5 hours of occlusion were reversible if 100% O2 inspiration was maintained during most of the occlusion. CONCLUSIONS: Ventilation with 100% O2 during occlusion increased the PO2 across most of the retina and partially restored the LERG b-wave, but the tissue near the vitreous was still sometimes anoxic. The illumination status seemed to make little difference. Inspiration of elevated O2 might be beneficial in treating retinal vascular occlusive disease, although it alone cannot completely maintain retinal function.

Oxygen distribution in the macaque retina.

PURPOSE: Oxygen distribution was characterized in the macaque retina, which is more like the human retina than others studied previously. METHODS: Profiles of oxygen tension (PO2) as a function of distance were recorded in a parafoveal region about halfway between the fovea and optic disk, and from the fovea in one animal. A one-dimensional diffusion model was used to determine photoreceptor oxygen consumption (QO2). RESULTS: In the parafovea, the PO2 decreased as the electrode was withdrawn from the choroid toward the inner retina, reaching a minimum value during dark adaptation of about 9 mmHg at about 70% retinal depth, and then increasing more proximally. Approximately 90% of the oxygen requirement of the photoreceptors was supplied by the choroidal circulation and 10% by the retinal circulation. In light adaptation, there was a monotonic PO2 gradient from the choroid to the inner retina, indicating that all of the oxygen used by photoreceptors was supplied by the choroid. In the fovea, the choroid supplied almost all the oxygen in both dark and light adaptation, with a minor supply from the vitreous humor. Dark-adapted foveal oxygen consumption was lower than parafoveal oxygen consumption. Light reduced the oxygen consumption of the photoreceptors, in both regions studied, by 16-36%. CONCLUSIONS: The results show that oxygenation of the parafoveal monkey retina is similar to that previously observed in the cat area centralis. In the fovea, the oxygen distribution differs as expected considering the thinner retina and the absence of inner retinal neurons and retinal circulation.

Oxygen consumption in the inner and outer retina of the cat.

PURPOSE: Oxygen consumption rate (QO2) was determined in the outer and inner halves of the cat retina in dark and light adaptation. METHODS: Double-barreled oxygen microelectrodes were used to measure oxygen tension (PO2) across the retina of anesthetized cats while the single retinal artery supplying that area was occluded. During the measurements of these PO2 profiles, the cats were ventilated with 100% O2. Retinal PO2 profiles were fitted to a diffusion model, and inner and outer retinal QO2s were determined from the fitted parameters. RESULTS: A four-layer model, in which two layers consumed oxygen, fitted the data well. One consuming layer corresponded to the photoreceptor inner segments, as in previous studies, and a single region of uniform consumption was used to describe the profile in the inner half of the retina. Under dark-adapted conditions, outer and inner retinal QO2 were 3.9 +/- 2.8 and 3.5 +/- 1.7 ml O2/(100 g.min) (mean +/- SD; 9 cats), respectively. With steady illumination, outer retinal (photoreceptor) QO2 decreased to 1.4 +/- 0.9 ml O2/(100 g.min), but inner retinal QO2 remained unchanged at 3.7 +/- 1.5 ml O2/(100 g.min) (5 cats). CONCLUSIONS: The total QO2 of the inner retina was found to be the same as that of the dark-adapted outer retina. Oxygen use was distributed uniformly throughout the inner retina but was confined to the photoreceptor inner segments, which occupied approximately 20% of the thickness of the outer retina. Steady illumination had no effect on inner retinal QO2.

Retinal hypoxia in long-term diabetic cats.

PURPOSE: To determine whether the retina is hypoxic in early stages of diabetic retinopathy in cats and to correlate intraretinal PO2 with fluorescein angiographic and histologic alterations. METHODS: Intraretinal PO2 was measured with microelectrodes in three cats with long-standing diabetes (>6 years) that had been followed with fluorescein angiographs every 6 months. Average PO2 in the inner vascularized half of the retina was compared with similar measurements in 21 control animals. Photoreceptor oxygen consumption was also compared. The retinal vascular endothelium of the diabetic animals was stained for ADPase activity in flatmounts, and transverse sections were used to visualize microscopic alterations in vascular structure. RESULTS: PO2 in the inner half of the retina was abnormally low in the diabetic cats, 7.7+/-5.2 mm Hg (35 penetrations in 3 cats) versus 16.4+/-9.3 mm Hg in normal cats (85 penetrations in 21 cats) (P << 0.001). Oxygenation was almost normal in some regions of the diabetic retinas, but little evidence of oxygen supply from the retinal circulation was observed in other regions. Inner retinal hypoxia was present in areas with no detectable capillary dropout in fluorescein angiograms or flatmounts. The worst changes histologically were microaneurysms, leukocyte and platelet plugging of aneurysms and venules, and degenerating endothelial cells in capillary walls. These histologic abnormalities were confined to small regions, some of which could be positively correlated with markedly abnormal PO2 profiles. Photoreceptor oxygen utilization was not affected in two diabetic cats, but was below normal in one animal in which choroidal PO2 was low. CONCLUSIONS: This is the first direct demonstration of retinal hypoxia in early diabetic retinopathy, before capillary dropout was evident clinically. Hypoxia was correlated with endothelial cell death, leukocyte plugging of vessels, and microaneurysms.

Local 42 degrees C hyperthermia improves vascular conductance of the R3230Ac rat mammary adenocarcinoma during sodium nitroprusside infusion.

The effect of sodium nitroprusside-induced hypotension on the perfusion of the R3230 adenocarcinoma during local 42 degrees C hyperthermia was studied using a combination of intravital microscopy and laser Doppler flowmetry. Fischer 344 rats were implanted with dorsal skin flap window chambers containing the R3230Ac tumor and allocated to three treatment groups (34 degrees C with nitroprusside, 42 degrees C with nitroprusside, and 42 degrees C with 0.9% saline). After baseline observation at 34 degrees C, tumors were locally heated to 42 degrees C using a water bath and either 0.9% saline or nitroprusside sufficient to reduce blood pressure 20% below pretreatment baseline was infused. Nitroprusside at 34 degrees C decreased tumor vascular conductance 40% with no effect on the diameter of arterioles entering the tumor. The diameter of arterioles entering 42 degrees C heated tumors increased 35% independent of blood pressure change. Saline at 42 degrees C had no effect on tumor vascular conductance; however, nitroprusside at 42 degrees C increased tumor vascular conductance 55%. Local 42 degrees C tumor heating, combined with a moderate reduction in blood pressure with nitroprusside, overrides the vascular steal effect associated with reduced perfusion pressure alone and results in improved tumor perfusion. Observations of the effect of vasodilator substances on normothermic tumor perfusion cannot be extrapolated to situations where moderate hyperthermia is used.

Effects of the interaction between carbogen and nicotinamide on R3230 Ac tumor blood flow in Fischer 344 rats.

Braun, R. D., Lanzen, J. L., Turnage, J. A., Rosner, G. and Dewhirst, M. W. Effects of the Interaction between Carbogen and Nicotinamide on R3230 Ac Tumor Blood Flow in Fischer 344 Rats. Radiat. Res. 155, 724-733 (2001). The purpose of this study was to determine whether there are interactions between carbogen breathing and various doses of nicotinamide at the level of the tumor arteriole that might contribute to the improvement in tumor blood flow and pO(2) that is often seen with this combination treatment. R3230 adenocarcinomas were implanted and grown to 4-5 mm in dorsal skin flap window chambers in F344 rats. Saline or 65, 200 or 500 mg/kg nicotinamide was injected i.p. while the rat breathed air through a face mask. After 20 min, either the breathing gas was switched to carbogen for 60 min or the animal remained on air. Measured end points included diameter of tumor arterioles, tumor perfusion, mean arterial blood pressure, and heart rate. None of the measured parameters were affected by injection of saline or nicotinamide, except at the highest nicotinamide dose (500 mg/kg). Mean arterial blood pressure showed a median decrease of 25% when 500 mg/kg nicotinamide was given. Diameter of tumor arterioles decreased significantly from 5-15 min after 500 mg/kg nicotinamide was given but was back to baseline by 20 min. Blood flow decreased significantly 5-20 min after administration of 500 mg/kg nicotinamide compared to the baseline prior to injection. Carbogen breathing resulted in a small increase in mean arterial blood pressure in all groups. There was a transient decrease in the diameter of tumor arterioles and blood flow during the first 5 min of carbogen breathing that was statistically significant in several groups. In the group injected with 500 mg/kg nicotinamide, the diameter of tumor arterioles increased by about 10% during the first 25 min of carbogen breathing, and blood flow increased by a median of 75% over the level prior to carbogen breathing up to 40 min after carbogen breathing. The increase in flow in this group was most likely caused by the concomitant arteriolar vasodilation. Thus there was direct evidence for an interaction between carbogen breathing and nicotinamide, but only at the dose of 500 mg/kg nicotinamide. Since this dose yields plasma levels of nicotinamide that are higher than can be tolerated clinically, it is uncertain whether these changes in arteriolar diameter and blood flow would occur in human tumors.

Stroma-free human hemoglobin A decreases R3230Ac rat mammary adenocarcinoma blood flow and oxygen partial pressure.

We examined the effect of a nitric oxide (NO) quencher, stroma-free human hemoglobin A (HbA0; 0.01, 0.05, 0.1, 0.2 g/kg), on the blood flow measured using the Doppler flow technique, tumor oxygen pressure (pO2) and the diameter of the arterioles using R3230Ac mammary adenocarcinoma as the tumor model. In female Fischer 344 rats with 1-cm-diameter tumors implanted in the lateral aspect of the left quadriceps, intravenous infusion of 0.1 and 0.2 g/kg HbA0 decreased both central tumor and peripheral tumor blood flow by 20-30% (P < 0.05). Tumor pO2 decreased 28% with 0.2 g/kg HbA0, from 15 mm Hg (baseline) to 11 mm Hg at 10 min (P = 0.02). Although 0.2 g/kg HbA0 increased blood flow 55% in the left quadriceps muscle proximal to the implanted tumor (P < 0.05), HbA0 had little effect on blood flow in right quadriceps muscle with no tumor implanted, and increased right quadriceps pO2, from 21 mm Hg (baseline) to 23 mm Hg at 10 min (P = 0.03). HbA0 increased mean arterial pressure 5-10% in a manner that was dependent on dose while heart rate concurrently decreased 9-19%. The diameter of the arterioles supplying the tumor was rapidly reduced 10% by 0.2 g/kg HbA0 (P = 0.037) and remained stable through 60 min of observation (P = 0.005). HbA0 selectively reduces tumor blood flow and tumor pO2 through vasoconstriction of the arterioles supplying the tumor. Vascular NO quenching provides an alternative to NO synthase inhibition as a means to achieve the goal of selective tumor hypoxia.

New perfluorocarbon emulsion improves tissue oxygenation in cat retina.

We have studied the conditions under which a perfluorocarbon emulsion of perfluorooctyl bromide (PFOB; Alliance Pharmaceutical, San Diego, CA) enhances tissue O2 delivery. Measurements of retinal tissue O2 tension (PO2) were made in anesthetized, artificially respirated, dark-adapted, normovolemic cats before, during, and after the infusion of three successive doses of 1 g PFOB/kg body wt each. There was little immediate effect of the infusion on the tissue PO2 when the cats were breathing room air, but the mean increase in tissue PO2 during 100% O2 breathing was 60 +/- 9% (SE; n = 8 cats) greater after infusion of 1 g PFOB/kg and approximately 136% greater after 3 g PFOB/kg. Similar infusions of the emulsifying medium alone had negligible effects on tissue PO2. These results suggest that PFOB emulsion may be clinically useful in treating tissue hypoxia in normovolemic patients breathing O2-enriched air.

Analysis of the heterogeneity of pO2 dynamics during photodynamic therapy with verteporfin.

Photodynamic therapy (PDT) with verteporfin provides a reliable way to destroy malignant tissues. Changes in the blood flow and oxygen partial pressure (pO2) during verteporfin-PDT were studied here in the tumor tissue of the rat mammary R3230Ac carcinoma model. Oxygen microelectrodes (6-12 microns tip diameter) were used to measure the transients locally within tumors during intravenous injection of 1.0 mg/kg verteporfin followed by irradiation 15 min later with 690 nm light at 200 mW/cm2, for a cumulative dose of 144 J/cm2. The observed changes in pO2 were heterogeneous and there was a difference in the response of low-pO2 regions relative to higher-pO2 regions. The change in pO2 in hypoxic tissue regions (pO2 < 8 mmHg) had acute pO2 loss after treatment, whereas the response in regions of higher pO2 (> 8 mm Hg) was more heterogeneous with some areas maintaining their pO2 value after treatment was completed. Blood flow measurements taken on a subset of the animals indicated a significant loss in flow during the initial light delivery that remained low after treatment, indicating some vascular stasis. The results suggest that hypoxic or poorly perfused vessels may be more susceptible to acute stasis than normoxic vessels in this treatment protocol.

Solubility of iron(II) carbonate at temperatures between 30 and 80 degrees.

Measurements were made of the forward rate-constant (k(f)) for the dissolution of FeCO(3) at 10 degrees temperature intervals between 30 and 80 degrees and in buffered solutions at pH 4, 5, 6 and 7. The solubility product (K(sp)) of FeCO(3) was measured at the same six temperatures. The forward rate-constant is related to temperature (T, degrees C) and pH by pk(f) = pH - 0.0350T + 0.695. The solubility product of FeCO(3) is related to temperature by pK(sp) = 0.0314T + 10.20. Kinetic data indicate that, under the conditions of the study, the rate-determining step of the dissolution reaction is FeCO(3)(s) + H(+) --> Fe(2+) + HCO(-)(3).