A comparative study of tumour blood flow modification in two rat tumour systems using endothelin-1 and angiotensin II: influence of tumour size on angiotensin II response.

Tumour blood flow modification following i.v. administration of angiotensin II (AT II, 0.19 nmol kg-1 min-1) or endothelin-1 (ET-1, 1 nmol kg-1) was compared in the P22 carcinosarcoma-bearing BD9 rat and the HSN fibrosarcoma-bearing CBH/CBi rat using the tissue uptake of radiolabelled iodoantipyrine. Results were compared with a range of normal tissues. HSN tumour blood flow was unmodified by either peptide, whereas P22 tumour blood flow was unmodified by ET-1 but was reduced to 80% of the control flow by AT II. Both peptides reduced absolute blood flow in the skin overlying the tumour, in contralateral skin, skeletal muscle, kidney and small intestine, whereas blood flow to the brain and heart was significantly increased by ET-1 and unmodified by AT II. Both peptides significantly increased vascular resistance (mean arterial blood pressure / tissue blood flow) in all normal tissues and both tumours, thus demonstrating the existence of vascular receptors for these 2 vasomodifiers, and the capacity of the vessels to respond to receptor activation. Dependency of response on tumour size was examined in the P22 tumour. In contrast to that in small P22 tumours (1.22 +/- 0.06 g), blood flow to large P22 tumours (7.18 +/- 0.25 g) was unmodified by AT II. Vascular resistance was equally increased in both tumour groups, thus illustrating little difference in the vascular response to AT II in the size range examined. Results show that the 2 rat tumours responded directly to ET-1 and AT II, but do not indicate any advantage of ET-1 over AT II in tumour blood flow modification. However, the existence of tumour vascular endothelin receptors suggests that the advent of less toxic and more controllable receptor ligands may make endothelin receptors of value in the modification of tumour blood flow.

Spatial heterogeneity of tumour blood flow modification induced by angiotensin II: relationship to receptor distribution.

Angiotensin II (ATII) has potential for improving delivery of blood-borne anti-cancer agents to tumours by increasing tumour blood flow. However, ATII-induced hypertension is not always accompanied by an increase in tumour blood flow due to significant constriction of the tumour vasculature. Such unpredictability in tumour response to ATII limits the clinical usefulness of this approach. In this study, the potential of assessing numbers of binding sites for ATII as a predictor of tumour blood flow response to intravenous administration of ATII was investigated. The distribution of ATII receptors in the rat P22 carcinosarcoma was related to tumour blood flow distribution and blood flow response to ATII using an autoradiographic approach. ATII (0.2 microgram x kg-1 x min-1) increased mean arterial blood pressure of anaesthetized BD9 rats from 92.2 +/- 1.4 mmHG to 145.6+- 1.3 mmHg. Despite this increase in perfusion pressure, overall tumour blood flow to viable regions decreased by 20%, indicating significant constriction of tumour blood vessels. Autoradiographic localisation of tumour blood flow showed that the decrease in flow was confined to the tumour periphery, with no change at the tumour centre. This pattern was consistent with 10% more binding sites for ATII at the tumour periphery than at the tumour centre. Maximum number of binding sites (BLmax) for the P22 tumour was 0.38 +/- 0.09 fmol x mg-1, which is approximately a factor of 10 lower than published values for various normal tissues. The dissociation constant Kd was l.16 +/- 0.18 nM. These results encourage the development of techniques for analysis of receptor binding characteristics for predicting the response of individual tumours to blood flow manipulation using vasoactive agents.

The response of tumour vasculature to angiotensin II revealed by its systemic and local administration to 'tissue-isolated' tumours.

A tissue-isolated preparation of the P22 rat carcinosarcoma was used to investigate the tumour vascular response to angiotensin II (ATII). In particular, the relative importance of systemic and local tumour factors was assessed by comparing tumour vascular resistance during systemic administration of ATII and during administration directly into the tumour-supplying artery. The effect of hypervolaemia on tumour vascular resistance was determined as well as the effect of ATII on oxygen metabolism. Tumour vascular resistance was increased by ATII in a dose-dependent manner. The response was biphasic with an initial peak in resistance followed by a lower plateau phase. Systemic administration of ATII was more effective in increasing tumour vascular resistance than direct administration. This suggests that systemic administration is not causing any reopening of previously collapsed tumour blood vessels. Further evidence for this is that hypervolaemia caused no reduction in tumour vascular resistance and that there was no difference in oxygen extraction by tumours between groups treated with systemically and directly administered ATII. A heterogeneous distribution of ATII receptors in the P22 tumour is a more likely explanation for the known heterogeneity of blood flow response to ATII.

Characterisation of tumour blood flow using a 'tissue-isolated' preparation.

Tumour blood flow was characterised in a 'tissue-isolated' rat tumour model, in which the vascular supply is derived from a single artery and vein. Tumours were perfused in situ and blood flow was calculated from simultaneous measurement of (1) venous outflow from the tumour and (2) uptake into the tumour of radiolabelled iodo-antipyrine (IAP). Comparison of results from the two measurements enabled assessment of the amount of blood 'shunted' through the tumours with minimal exchange between blood and tissue. Kinetics of IAP uptake were also used to determine the apparent volume of distribution (VDapp) for the tracer and the equilibrium tissue-blood partition coefficient (lambda). lambda was also measured by in vitro techniques and checks were made for binding and metabolism of IAP using high-pressure liquid chromatography. VDapp and lambda were used to calculate the perfused fraction (alpha) of the tumours. Tumour blood flow, as measured by IAP (TBFIAP), was 94.8 +/- 4.4% of the blood flow as measured by venous outflow, indicating only a small amount of non-exchanging flow. This level of shunting is lower than some previous estimates in which the percentage tumour entrapment of microspheres was used. The unperfused fraction ranged from 0 to 20% of the tumour volume in the majority of tumours. This could be due to tumour necrosis and/or acutely ischaemic tumour regions. For practical purposes, measurement of the total venous outflow of tumours is a reasonable measure of exchangeable tumour blood flow in this system and allows for on-line measurements. Tracer methods can be used to obtain additional information on the distribution of blood flow within tumours.

Resistance to flow through tissue-isolated transplanted rat tumours located in two different sites.

The perfusion characteristics of the P22 carcinosarcoma were investigated in tissue-isolated tumour preparations in the ovarian and inguinal fat pads of BD9 rats. Tumours were perfused with a physiological buffer of known viscosity and changes in perfusion pressure were recorded at different perfusion rates in an ex vivo system. At perfusion pressures exceeding 30-40 mmHg tumour flow rate was directly proportional to the perfusion pressure in all tumours, indicating a constant resistance to flow. An apparent positive pressure difference across the tumour vasculature of 20-30 mmHg occurred under conditions of zero flow in either site. At low perfusion pressures, the flow resistance increased sharply due to increases in the geometric resistance of the tumours. These findings are in accord with previously published data. Geometric resistance increased with tumour volume in both sites and was approximately five times greater in the inguinal tumours than it was in the ovarian tumours, on a weight to weight basis. The dependence of tumour geometric resistance on perfusion pressure differs from the situation in normal tissues and may provide a means of manipulating the tumour microcirculation to the exclusion of the systemic blood supply. The dependence of geometric resistance on tumour site may partly explain why tumours located in different sites respond differently to various forms of therapy.

Modification of tumour blood flow using the hypertensive agent, angiotensin II.

The effects of different doses of angiotensin II (0.02 to 0.5 microgram kg-1 min-1 on mean arterial blood pressure, tissue blood flow and tissue vascular resistance were investigated in BD9 rats. Blood flow was measured using the uptake of 125I- or 14C-labelled iodoantipyrine (125I-IAP and 14C-IAP). Spatial heterogeneity of blood flow within tumours, before and after angiotensin II infusion, was also measured using 14C-IAP and an autoradiographic procedure. Mean arterial blood pressure rose steeply with angiotensin II dose. Blood flow to skeletal muscle, skin overlying the tumour, contralateral skin, small intestine and kidney tended to decline in a dose-dependent manner. Blood flow to the tumour was also reduced (to 80% of control values) but there was no dose response. Blood flow to the heart was slightly increased and blood flow to the brain was unaffected by angiotensin II. Vascular resistance, in all tissues, was increased by angiotensin II infusion. The increase in tumour tissue was similar to that found in skeletal muscle and small intestine and is likely to be caused by a direct vasoconstricting effect of the drug rather than autoregulation of tumour blood flow in the face of an increase in perfusion pressure. The reduction in overall blood flow at the highest perfusion pressure was due to a preferential effect of angiotensin II at the tumour periphery. These results show that some tumours, at least, can respond directly to the effects of vasoactive agents.

Changes in tumour morphology with alterations in oxygen availability: further evidence for oxygen as a limiting substrate.

The ability of cancer cells to survive at a distance from blood vessels should be dependent on the local supply of nutrients to each vessel. The corded growth of tumour cells around blood vessels within regions of necrosis in the RH carcinoma in the mouse allows the limit to which cells can be supported by individual vessels to be observed. The thickness of individual tumour cords was measured in conventionally stained tumour sections using a scanning technique to determine the distance between the blood vessel wall and the most distant viable cell adjacent to necrosis. Cord radius was found to vary with the oxygen supply conditions. Control animals had a mean radius of 105 +/- 2 microns while animals that had breathed 10% oxygen had significantly narrower cords (93 +/- 3 microns after 48 h) and animals breathing 100% oxygen had significantly wider cords (117 +/- 3 microns after 24 h). Mice made anaemic (mean hct. 28%) by phlebotomy and plasma transfusion had cord radii that were not significantly different from controls at any time up to 48 h. We conclude that this relatively slow growing mouse tumour is capable of rapid morphological adaptation (less than 3 h) to changes in nutrient availability and that oxygen is probably the limiting substrate.