[CANCER RESEARCH 38, 2385-2390, August 1978]0008-5472/78/0038-0000$02.00
Effects of Several Vasoactive Drugs on the Vascular Resistance ofMT-W9B Tumors in W/Fu Rats1
Randy Jirtle,2 Kelly H. Clifton, and John H. G. Rankin3
Radiobiology Laboratories, Wisconsin Clinical Cancer Center, and Departments of Human Oncology and Radiology [R. J., K. H. C.] and Physiology andGynecology-Obstetrics ¡J.H. G. R.¡,University of Wisconsin Medical School, Madison 53706, and Wisconsin Perinatal Center, Madison General Hospital [J.H. G. R.], Madison, Wisconsin 53715
ABSTRACT
These experiments were designed to study the effectsof vasoactive drugs on normal and malignant tissue in W/Fu rats. The increase in resistance to tumor blood flowelicited by a bolus injection of 10 jug of norepinephrinewas greater than that elicited in the surrounding mammary gland tissue. A 10-fold increase in the resistance totumor blood flow was sustained for 30 min by the infusionof norepinephrine at the rate of 1.39 /¿g/min,whereas asmaller initial increase in mammary gland vascular resistance decreased with time. In contrast, the increase inresistance to tumor blood flow caused by a bolus injectionof angiotensin II was less than that observed in themammary gland tissue. A 20-fold increase in mammarygland vascular resistance could be maintained for at least5 min by infusion of angiotensin II at the rate of 1.39 ¿ig/min. In comparison, such treatment caused only a 3-foldincrease in the resistance to tumor blood flow. A bolusinjection of 1 ng of isoproterenol decreased the vascularresistance in all normal tissues studied, but the resistanceto blood flow in the tumor remained unchanged. Theresults of these experiments indicate that there may bemethods whereby the tumor blood flow can be manipulated for therapeutic purposes and to assist radiographievisualization of tumors.
INTRODUCTION
The literature concerning the response of tumor vascula-ture to vasoactive drugs is often contradictory. For exampleit has been reported that tumors respond poorly to epineph-rine when compared to responses of the surrounding tissue(1, 22), that the arterioles supplying a tumor respond toepinephrine in a manner similar to that of normal somaticvessels distant from the tumor (11), and that changes intumor blood flow secondary to epinephrine infusion aremore pronounced than those in the surrounding normaltissue (7).
Much of this conflict arises because of variations intumor-host systems, anesthetic techniques, the state of thepreparation, and the techniques used for measuring bloodflow. We have recently shown that radioactive microspherescan be used to measure blood flow to the V2 carcinoma inthe rabbit (16). In these studies we have found that the
1 Supported by NIH and National Cancer Institute Grant CA18756, and by
the American Cancer Society Grant PDT-46R.2 Supported in part by a grant from the Graduate School of the University
of Wisconsin, Madison, Wis.3 To whom requests for reprints should be addressed, at Room 7224 East,
Madison General Hospital, 202 South Park Street, Madison, Wis. 53715.Received October 11, 1977; accepted April 19, 1978.
increase in resistance to tumor blood flow after the injectionof norepinephrine was greater than that of the surroundingnormal tissue. The rabbit preparation suffered from thedisadvantage that the foreign tumor material produced animmune reaction in the surrounding tissue which confusedthe results. We have determined that a superior preparationcan be obtained with the use of the inbred W/Fu rat carryingthe MT-W9B mammary tumor. We have previously demonstrated that it is possible to measure tumor blood flow twicein an unanesthetized rat with 2 injections of microspherescontaining different radioactive labels (10). In the experiments reported here, we have used that preparation toexamine the effects of various vasoactive drugs on theresistance to blood flow in normal and malignant tissues.
MATERIALS AND METHODS
Animal and Tumor System. Female isogeneic W/Fu ratsweighing approximately 240 g were housed 2 to a suspended cage in a temperature-controlled room with 12 hrof light daily. Food and water were given ad libitum.
Tumor suspensions were prepared for transplantationwith a Snell cytosieve as previously described (5) and wereadjusted to a 33% volume of centrifugally packed cellularmaterial from an MT-W9B mammary adenocarcinoma (11).Inocula of 0.10 ml were injected into both axillary mammaryglands and the right inguinal mammary gland.
Surgical Procedure. The animals were anesthetized withether, and catheters were surgically placed into both theleft ventricle of the heart and the left femoral artery whentumors reached approximately 1 g. The animals were returned to cages and allowed to recover for approximately 3hr before the experiments were performed. Full details ofthese procedures are described elsewhere (10).
Microsphere Technique. Approximately 70,000 radioac-tively labeled microspheres were slowly flushed into the leftventricle of the heart with 0.6 ml of 0.9% NaCI solution. AHarvard withdrawal pump was used to withdraw simultaneously a femoral arterial blood sample for 1 min at a rate of0.51 ml/min. For reasons previously described, all tissueblood flows were estimated with spheres 25 /¿min diameter(10). The spheres were labeled with either 125I,46Sc, 85Sr,141Ce(Minnesota Mining and Manufacturing Co., St. Paul,Minn.), 54Mn, or 109Cd (New England Nuclear, Boston,
Mass.).Approximately 15 min later, 0.1 ml of various vasoactive
drugs of differing concentrations was flushed into the leftventricle of the heart with 0.5 ml of 0.9% NaCI solution.After 0.75 min, 25-/¿mmicrospheres, labeled with an isotope with a characteristic y spectrum that differed from thatof the first, were flushed into the left ventricle of the heart
with 0.6 ml of 0.9% NaCI solution. An integrated arterialblood sample was again withdrawn. In 1 series of experiments, the second batch of microspheres was injectedeither 0.75, 2, or 5 min after a bolus injection of 10 /¿gofnorepinephrine. In another series of experiments, eithernorepinephrine or angiotensin II was infused into the rightjugular vein at a rate of 1.39 /¿g/minfor various lengths oftime before the second batch of labeled microspheres wasinjected.
The animals were sacrificed by the injection of 0.10 ml ofeuthanasia solution (Vet Labs, Lenexa, Kans.) into the leftventricle of the heart. The skin from the right inguinalregion, the right inguinal mammary gland, the lower hindleg muscle, and the tumors were dissected free of surrounding extraneous tissue and were individually placedinto glass counting vials. For each tissue sample the 2isotope activities and the corresponding number of sphereswere determined by appropriate data reduction of theoutput from a 3-channel Nal well counter equipped withpulse height analyzers (17).
The rationale for our use of microspheres to measuretumor blood flow is as follows. The microsphere method isthe only method that permits the simultaneous measurement of blood flows to many regions in the same animal(12, 18). Buckberg ef al. (4) have evaluated the method andhave reported on the potential sources of error. The methodhas been used in rats by many investigators (10,13,14, 21).Nishiyama ef al. (14), using microspheres, measured theregional distribution of cardiac output in unanesthetizedrats. McDevitt and Nies (13) have also reported that micro-spheres can be used to estimate the cardiac output anddistribution in rats, provided that the blood samples containmore than 400 spheres. They reported that the injectioncaused no hemodynamic changes and that the resultsagree with published estimates calculated by other techniques. Tsuchiya ef al. (21) have used microspheres inunanesthetized rats and reported that there were no hemodynamic alterations and that the reproducibility of 3 separate injections to each'rat was excellent. We have previously
used microspheres to measure the blood flow to the V2carcinoma in rabbits (16), and we have performed extensiveexperiments on the use of this technique for the measurement of mammary tumor blood flow in unanesthetized rats(10). We conclude that microspheres can be used to measure regional blood flows in unanesthetized rats.
Resistance Ratio Calculations and Statistical Analysis.The tissue blood flows were calculated by the equation
FT = (WR/NB) x NT
where FT is the tissue blood flow (ml/min), WR is thewithdrawal rate of the integrated arterial blood sample, N„is the number of spheres in the withdrawn blood sample,and NT is the number of spheres in the sample tissue. Thesystemic arterial blood pressure was measured with aStatham pressure transducer and recorded on chart paper.Since the pressure could not be monitored when blood wasbeing withdrawn from the femoral artery, the average arterial blood pressure during this time was estimated by linearinterpolation. On the assumption that the mean venouspressure is equal to zero, the tissue vascular resistance
(mm Hg/ml/min/g) could be calculated by the equation
RT= Pal Fr
where Pa is the average systemic arterial blood pressure(mm Hg).
The tissue blood flows and systemic arterial pressureswere estimated before and after the injection of vasoactivedrugs, which allowed calculation of the resistance to bloodflow in the various tissues at these times. The ratio of thetissue blood flow resistance after the injection of a vasoactive drug to that before injection (i.e., resistance ratio =treatment/control) yields an estimate of the fractionalchange in tissue vascular resistance caused by the drug.Therefore, if 0.9% NaCI solution is injected rather thanvasoactive drugs, theoretically the resistance ratio shouldbe 1.0. However, with this animal system, the resistanceratios, under this condition, for skin, muscle, mammarygland, and tumors were previously determined to be 1.61,1.24, 1.60, and 1.90, respectively (10).
The statistical procedure, ANOVA, was used to compare2 or more independent sample means, and the paired t testwas used to compare both the tumor blood flows andresistance ratios to the corresponding values in the variousnormal tissues. A x2 test was used to determine whether the
distribution of the resistance ratios was adequately described by a normal probability density function (3).
Drugs. Norepinephrine (Levophed bitartrate, WinthropLaboratories, New York, N. Y.), angiotensin II (Bachern,Inc., Marina del Rey, Calif.) and isoproterenol (Isuprel hy-drochlonde; Winthrop Laboratories) were all diluted to theappropriate concentration in 0.9% NaCI solution and werealways stored in the frozen state to minimize deterioraron.
RESULTS
Frequency Distribution of the Resistance Ratio Data. AX2test demonstrated that the distribution of the resistance
ratios (i.e., treatment/control) could not be described adequately by a normal probability density function (p < 0.001).The blood flow and the resistance ratio distributions could,however, be normalized with a natural logarithmic (In)transformation of the data and all parametric statisticaltests (e.g., ANOVA, and f tests) were therefore performedafter In transformations.
Effect of Norepinephrine. The ratio of vascular resistances (i.e., treatment/control) as a function of the injecteddose of norepinephrine for normal and malignant tissues isshown in Chart 1. A bolus injection of 5 ¿igof norepinephrine produced a maximum response in the mammary gland,the muscle, and the skin. In the tumor, more than 5 ^g ofnorepinephrine were required to maximize the resistanceratio (p < 0.05). The average resistance ratios for themalignant tissue, after bolus injections of 10 or 20 /¿gofnorepinephrine, were significantly larger than those of thenormal surrounding mammary gland tissue (p < 0.01).
The increase in vascular resistance after a bolus injectionof 10 ju.gof norepinephrine was maintained longer in themalignant tissue than in the surrounding normal mammarygland tissue (i.e., 2 min, p < 0.001). After 5 min the resist-
Chart 1. Average resistance ratios (treatment/control) for muscle (A), skin(O), mammary gland (•).and MT-W9B mammary adenocarcinomas (D) as afunction of dose of norepinephnne 0.75 min after a bolus injection. Standarderrors of the means at each dose are of similar magnitude for the normaltissues, and only those for the mammary gland and the MT-W9B tumor arepresented. The numbers of observations for all normal tissues at each doseare identical, and those for the mammary gland and the MT-W9B tumor areprovided in parentheses.
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Chart 2. Average resistance ratios (treatment/control) for muscle (A), skin(O), mammary gland (•),and MT-W9B mammary adenocarcinomas (D) as afunction of time after a bolus ¡ntraarterialinjection of 10 M9 of norepinephrine. Standard errors of the means at each dose are of similar magnitudefor the normal tissues, and only those for the mammary gland and the MT-W9B tumor are presented. The numbers of observations for all normaltissues at each time are identical, and those for the mammary gland and theMT-W9B tumor are provided in parentheses.
ance to blood flow in all the tissues studied had returned tocontrol levels (Chart 2).
When norepinephrine was infused into the jugular vein ata rate of 1.39 ¿ig/min,the resistance ratio for the tumortissue growing in the mammary glands was significantlygreater than the control value of 1.90 (95% confidenceinterval, 1.14 to 3.18) and was significantly larger than thatof all normal tissues studied for the entire 30-min infusionperiod (Chart 3a). The vasculature of the mammary gland
Effects of Drugs on Rat Tumor Vascular Resistance
partially escaped from the effect of norepinephrine in 30min of infusion (p < 0.01) but did not return completely tothe control value. In contrast, the resistance ratio of thetumors growing in mammary gland tissue did not significantly change during the 30 min of infusion. During norepinephrine infusion the relative blood flow to the tumor wasinitially 4 times that of the surrounding tissue (Chart 30).The blood flows to the tumor and surrounding tissues werealmost identical after 15 min of infusion. Compared to skinand mammary gland, muscle tissue, although sensitive tobolus injections of norepinephrine, was relatively unresponsive to its infusion at the rate used.
Effect of Angiotensin II. In contrast to the results obtained with norepinephrine, we found that a bolus injectionof angiotensin II elicited a smaller resistance ratio increasein the tumor than in the mammary gland (Chart 4). Similarresults were obtained when angiotensin II was infused at arate of 1.39 /¿g/min(Chart 5a). Short-term constant infusionstudies also demonstrated that the vasculatures of the
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Chart 3. Average resistance ratios [treatment/control (a) and blood flows(£>)]for muscle (A), skin (O), mammary gland (•),and approximately 1 gMT-W9B mammary adenocarcinomas (D) as a function of time after theinitiation of a continuous i.v. infusion of norepinephrine at a rate of 1.39 ng/min. Standard errors for the mean resistance ratios at each time are ofsimilar magnitude for the normal tissues, and only those for the mammarygland and the MT-W9B tumor are presented. Standard errors for the mean
blood flows are presented if larger than the symbol. The numbers ofobservations at each time for all normal tissues are identical, and those forthe mammary gland and the MT-W9B tumor are provided in parentheses.
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Chart 4. Average resistance ratios (treatment/control) for muscle (A), skin( O), mammary gland (•),and MT-W9B mammary adenocarcinomas (D) as afunction of dose of angiotensin II 0.75 min after a bolus injection. Standarderrors of the means at each dose are presented if larger than the symbol.The numbers of observations for all normal tissues at each dose areidentical, and those for the mammary gland and the MT-W9B tumor areprovided in parentheses.
mammary gland and of the skin were unable to escapesignificantly from the constricting effect of the drug for atleast 5 min. During infusion the blood flow to the skin andmammary gland dropped approximately 93%, whereas thatto the tumor decreased only 35% (Chart 50). Muscle tissuedid not respond significantly to either bolus injections (20/¿g)or constant infusions of angiotensin II. However, therelative blood flow increased during the infusion (Chart 5)because the systemic arterial pressure increased from 91 ±5 (N = 9) to 142 ±7 (N = 9) mm Hg.
Effect of Isoproterenol. After a bolus injection of 1 ¿¿gisoproterenol, the resistance ratios of all normal tissueswere reduced when compared to those of the controls (p <0.05; Table 1). Of the normal tissues studied, the vascula-ture of the skeletal muscle was the most responsive, andthe blood flow increased from 0.271 (95% confidence interval, 0.217 to 0.320) to 0.526 (95% confidence interval, 0.386to 0.950) ml/min/g (p < 0.001). In contrast, the resistanceto tumor blood flow did not change although the relativeblood flow was reduced by 55 ±5% (N = 21) due to adecrease in the average systemic arterial blood pressurefrom 96.8 ±2.3 (N = 125) to 79.7 ±3.9 (N = 14).
DISCUSSION
The microsphere technique enabled us to measure tissueblood flows in conscious, minimally disturbed rats beforeand after an exogenous administration of a vasoactive drug.
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Chart 5. Average resistance ratios [treatment/control (a) and blood flows(£>))for muscle (A), skin (O), mammary gland (•),and approximately 1 gMT-W9B mammary adenocarcinomas (D) as a function of time after theinitiation of a continuous i.v. infusion of angiotensin II at a rate of 1.39 ¿¿g/min. Standard errors of the means are shown If larger than the symbol. Thenumbers of observations for all normal tissues at each time are identical,and those for the mammary gland and the MT-W9B tumor are provided inparentheses.
Table 1Effect of a bolus injection of 1 ftg of isoproterenol on the vascular
resistance in various tissues of tumor-bearing rats
" Numbers in parentheses, 95% confidence interval.
Since the average systemic arterial blood pressure was alsomonitored, the magnitude of the changes in resistance totissue blood flows could be estimated. The microspheretechnique, however, does not allow one to determine theposition in the vascular network (i.e., preexisting normaltissue vasculature and/or newly formed tumor vasculature)where the change in resistance to tumor blood flow occurred. We have thus avoided the term "response of tumorvasculature" throughout. The results of these studies indi
cate that the change in the resistance to tumor blood flowcaused by the administration of norepinephrine, angiotensin II, or isoproterenol was of a different magnitude thanthat of the surrounding normal tissue. These differencesmay be exploitable to improve the treatment and diagnosisof certain tumors.
Bolus injections of norepinephrine at all doses studiedcaused an increase in the resistance to blood flow in bothnormal and malignant tissue. The resistance ratio for normal tissue was maximal after 5 /¿g,but malignant tissuerequired more than 10 jug. The maximal response observedin the malignant tissue was, however, significantly largerthan that of all normal tissues studied including the mammary gland, the normal tissue from which the tumor vasculature was derived (2) (Chart 1). Edlich ef al. (7) observedsimilar results when they compared the vascular responseof the amelanotic melanoma to that of the surrounding skinand muscle after a systemic administration of norepinephrine.
We also observed that the resistance ratio remainedelevated for a longer period of time in the tumor tissue thanin the surrounding normal tissue, which implied that themalignant tissue did not possess the same ability to escapefrom the effect of norepinephrine. This conclusion wasconfirmed when norepinephrine was infused at a constantrate of 1.39 /¿g/min.The resistance ratio of the tumor tissuedid not decrease significantly for 30 min, whereas theresistance ratio of the mammary gland tissue decreasedtoward control.
A tumor weighing approximately 1 g was found to have arelative blood flow (ml/min/g) about 330% greater than thatof mammary gland tissue. After 15 min of constant infusion,both the mammary gland tissue and the overlying skin hadpartially escaped from the effect of the norepinephrine,whereas the malignant tissue had not. As a consequencethe relative tumor blood flow was only 10% greater thanthat of the mammary gland.
These results may have considerable practical importance. For example, Steckel ef al. (20) have proposed theuse of catecholamines in conjunction with radiotherapy toprotect the normal tissue at risk (e.g., intestine and kidney).Our results indicate that, if the tumor to be treated isgrowing in the normal tissue at risk, the infusion of norepinephrine will equally protect the malignant and surroundingnormal tissue since their blood flows will be reduced to asimilar level. If the tumor is not in the normal tissue at riskit would be preferable to infuse the vasoactive drugs at arate such that an insignificant dose is delivered to the tumorupon its recirculation.
Dickson ef al. (6) have shown that the temperature in theYoshida tumor heated in a radiofrequency field, was 2-3°
lower than that in the surrounding muscle. They concludedthat the blood flow to the tumor was greater than that to themuscle. We have shown that such a condition exists whenthe MT-W9B tumor is growing in the mammary gland. Ifsuch a phenomenon is shown to hold more generally, theeffectiveness of hyperthermia treatment would be severelycompromised. However, the constant infusion of norepi-nephrine significantly lowers the difference between theblood flow to the malignant tissue and the blood flow to thesurrounding normal tissue. This presumably would reducetemperature differences during hyperthermia therapy. Inaddition, since tumor blood flow can be significantly decreased for extended periods of time, the intratumoral pHwould presumably decrease to a level that is even lowerthan that which normally occurs (9), thus increasing thesensitivity of the malignant cell to hyperthermia treatment(15).
When angiotensin II was injected into tumor-bearing rats,the results were opposite to those resulting from norepi-nephrine. The increase in the resistance ratio for tumors atall doses was significantly less than that for the mammarygland tissue and skin. When infused at a rate of 1.39 HQ/min, angiotensin II reduced the blood flow to the tumor by35%, whereas the flow to the mammary gland and overlyingskin was decreased approximately 93%. Therefore, after theinitiation of angiotensin II infusion, the blood flow to thetumor is 3400% rather than 330% greater than that to thesurrounding normal mammary tissue. By the constant infusion of angiotensin II, this condition can be maintained forat least 5 min.
Recently, it has been shown that the therapeutic resultsachieved by cesium therapy of breast carcinomas comparefavorably with other modalities of treatment, and mutilatingsurgery was avoided in 80% of those patients who survived5 years (19). If human mammary gland tissue responds toangiotensin II like rat mammary tissue, our results implythat local infusion of angiotensin II may further improve theradiation treatment of mammary carcinomas by protectingthe normal mammary gland tissue from radiation damage.
Ekelund ef al. (8) have demonstrated that angiotensin IIimproved radiographie visualization of bone and soft tissuetumors more than did norepinephrine and tolazoline. Ourresults suggest that this probably is due to the fact that,unlike most normal tissues, the resistance to tumor bloodflow is not greatly increased with angiotensin II. We suggestthat, since the mammary gland vasculature is very sensitiveto angiotensin II, the radiographie resolution of mammarygland tumors may also be enhanced by a prior local injection of this drug.
When isoproterenol, a vasodilator, was injected into tumor-bearing animals, the resistance to blood flow decreased in all normal tissues studied. The vasculature ofskeletal muscle was particularly sensitive to its action, andthe blood flow almost doubled after a bolus injection of 1¿ig.The resistance to tumor blood flow, however, wasunchanged; but since the systemic arterial pressure decreased, the tumor blood flow also decreased. These results provide an explanation of why /3-agonists do notusually enhance the radiographie visualization of tumorssurrounded by skeletal muscle (8). In addition, they imply
that tumor vasculature is insensitive to this /3-receptoragonist and/or is maximally dilated.
The microsphere technique has enabled us to studysimultaneously the response of normal and malignant tissue to vasoactive drugs. The response of the tumor relativeto that of the surrounding normal tissue is dependent uponthe drug injected, its concentration, and the time after itsadministration. The reason(s) for these differences are notcurrently known; nevertheless the results indicate that theappropriate use of vasoactive drugs might well improveboth the therapy and radiographie visualization of tumors.
ACKNOWLEDGMENT
The authors are indebted to Terranee Phernetton for excellent technicalassistance.
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1978;38:2385-2390. Cancer Res Randy Jirtle, Kelly H. Clifton and John H. G. Rankin of MT-W9B Tumors in W/Fu RatsEffects of Several Vasoactive Drugs on the Vascular Resistance
