Edgardo T. Ong

Analysis of the effects of oxygen supply and demand on hypoxic fraction in tumors.

The extent of hypoxic regions in a tumor tissue depends on the arrangement, blood flow rate and blood oxygen content of microvessels, and on the tissues oxygen consumption rate. Here, the effects of blood flow rate, blood oxygen content and oxygen consumption on hypoxic fraction are simulated theoretically, for a region whose microvascular geometry was derived from observations of a transplanted mammary andenocarcinoma (R3230AC) in a rat dorsal skin flap preparation. In the control state, arterial PO2 is 100 mmHg, consumption rate is 2.4 cm3 O2/100 g/min, and hypoxic fraction (tissue with PO2 < 3 mmHg) is 30%. Hypoxia is abolished by a reduction in consumption rate of at least 30%, relative to control, or an increase in flow rate by a factor of 4 or more, or an increase in arterial PO2 by a factor of 11 or more. These results suggest that reducing oxygen consumption rate may be more effective than elevating blood flow or oxygen content as a method to reduce tumor hypoxia.

Quantification of longitudinal tissue pO2 gradients in window chamber tumours : impact on tumour hypoxia

SummaryWe 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 μm. 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 μm. 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 ~50 vs >200 μm 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.

Perivascular Oxygen Tensions in a Transplantable Mammary Tumor Growing in a Dorsal Flap Window Chamber

Fischer 344 rats with R3230 Ac mammary carcinomas implanted in dorsal flap window chambers served as a model to obtain measurements of perivascular and stromal oxygen tension in normal and tumor tissues using Whalen recessed-tip microelectrodes (3- to 6-microns tip). Perivascular measurements were made adjacent to vessels with continuous blood flow. Thus the measurements and models provided are reflective of conditions leading to chronic hypoxia. Perivascular oxygen tensions averaged 72 +/- 13 mmHg in normal tissue vessels adjacent to tumor, 26 +/- 5 mmHg in tumor periphery, and 12 +/- 3 mmHg in tumor central vessels. There was a significant trend toward lower perivascular oxygen tensions in the tumor center (Kruskal-Wallis test, P = 0.002). A similar tendency was seen with a limited number of stromal measurements. Krogh cylinder models, which incorporate these data for perivascular oxygen tension, along with morphometric data obtained from the same tumor model suggest that hypoxic regions will exist between tumor vessels in the tumor center unless O2 consumption rates are well below 0.6 ml/100 g/min. The low perivascular measurements observed near the tumor center combined with the theoretical considerations suggest, for this model at least, that tissue oxygenation may best be improved by increasing red cell velocity and input pO2 and reducing oxygen consumption. The low perivascular oxygen tensions observed near the center also suggest that conditions conducive to increased red cell rigidity exist, that drugs which can decrease red cell rigidity could improve tumor blood flow and oxygenation, and that the endothelium of those vessels may be susceptible to hypoxia-reoxygenation injury.

Simultaneous Measurement of Liposome Extravasation and Content Release in Tumors

Objective: The success of liposome‐based drug delivery systems for tumor targeting relies on maximum extravasation of liposomes into tumor intersritium, as well as optimal release of contents from the liposomes once within the tumor. Liposome extravasation and content release are two separate processes that can be individually or jointly manipulated, so a method is needed to monitor these two processes independendy and simultaneously. In this report, we describe a method to measure liposome extravasation and content release in tumor tissues growing in a rat skinfold window chamber preparation.

Nitric oxide synthase inhibition irreversibly decreases perfusion in the R3230Ac rat mammary adenocarcinoma

We examined the microvascular effects of competitive nitric oxide synthase (NOS) inhibition with NG-monomethyl-L-arginine (MeArg), followed by L-arginine, on R3230Ac mammary adenocarcinoma perfusion. In window preparations containing tumours, superfusion of 50 microM MeArg reduced diameters of central tumour venules by 13%, of peripheral tumour venules by 17% and of normal venules near tumours by 16% from baseline. MeArg reduced red blood cell (RBC) velocity in central tumour venules by 25%, and increased intermittent flow and stasis frequency by 20% in central tumour venules. Subsequent superfusion of 200 microM L-arginine did not restore diameters or RBC velocity of any tumour preparation venules, and decreased length density in both central tumour venules and peripheral tumour venules. In contrast, MeArg reduced control preparation venule diameter by 30% and RBC velocity by 66%, but did not decrease length density or increase intermittent flow or stasis frequency. Unlike tumour preparation venules, L-arginine restored control venule diameters and velocities. NOS inhibition reduces both tumour and control venule perfusion, but the effect is blunted in the vicinity of tumours, possibly because of increased NOS levels. Perfusion can be subsequently restored in control, but not tumour, venules with L-arginine. Tumour NOS inhibition, followed by normal tissue rescue with L-arginine, may provide a novel means to achieve the goal of selective tumour hypoxia.

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.