(CANCER RESEARCH56, 4728-4734. October 15, 1996]

and suggest strongly that low hemoglobin levels are an indicator ofpoor prognosis in patients undergoing radiotherapy. Consequently,homologous blood or RBC transfusion is given frequently to severelyanemic cancer patients prior to radiotherapy. Although several investigations have been undertaken to assess the effects of transfusion ontherapy outcome, the results from these clinical studies have notshown convincingly a significant improvement in response to radiotherapy (10). This failure may, in some cases, be explained by therecruitment of patients with already very advanced disease, but immunosuppression associated with homologous blood transfusion mustalso be considered as playing a detrimental role (11—13).In addition,recent focus on transfusion-related infection risks and on the development of hemosiderosis or alloantibodies has brought about a changein general attitudes toward homologous blood transfusion practice.These last two aspects could be overcome by an autologous bloodtransfusion. Indeed, the first results of a study in which tumor recur

rence after surgical treatment of colorectal cancer in patients receivingeither autologous or homologous blood transfusion was comparedwould tend to support this notion (14). However, the use of autologous blood transfusions cannot be considered as a generally feasibleapproach in already anemic tumor patients. An alternative to transfusion is the use of erythropoietin, a glycoprotein hormone regulatingthe differentiation and maturation of RBCs and preventing apoptosisof progenitor cells. This approach to the correction of malignancyassociated anemia has received even more attention since rhEPO4became available for clinical use. Recent publications have shown that

rhEPO can be used effectively and safely in patients to correct

malignancy-associated anemia (15—22).To date, however, no studiesare available in which the effects of rhEPO on a range of tumorpathophysiological factors have been investigated. In this study, ananimal model of tumor anemia was established, and the effects ofrhEPO application on RBC-related parameters and on tumor oxygenation, blood flow, and bioenergetic status were determined, to ascertam whether this hormonal approach causes modifications in theseparametersthatmighthaveconsequencesfor anemictumorpatientsinterms of therapy outcome.

MATERIALS AND METHODS

Animals

Male Sprague-Dawleyrats(CharlesRiver Wiga, Sulzfeld, Germany;bodyweight, 160—210g) housed in our animal care facility were used in this study.They received a standard diet and water ad libitum. Experiments were conducted after authorization by the competent ethics committee according toGerman federal law.

Drugs

rhEPO (Recormon, purity >98%; Boebringer Mannheim, Mannheim,Germany) was dissolved in buffered saline and administered (1000 lU/kg)three times per week, over 14 days, to animals by s.c. injection. Normal(control) animals received an equivalent volume of isotonic PBS. Studies in

4 The abbreviations used are: rhEPO, recombinant human erythropoietin; p02. oxygen

partial pressure; 502, oxyhemoglobin saturation; TBF, tumor blood flow; MABP, meanarterial blood pressure; cHb, hemoglobin concentration.

ABSTRACT

Growth, blood flow, oxygenation, and bioenergetic status of experimentel tumors were Investigated In normal (control) and anemic animals afteradministration of recombinant human erythropoietin (rhEPO). DS sarcomas were Implanted s.c. onto the hind foot dorsum of Sprague-Dawleyrats. Tumor-associated anemia was induced by the development of an i.p.hemorrhagic ascites. rhEPO (1000 lU/kg) was administered s.c. threetimes per week over 14 days, after which it was found to have significantlyincreased hematocrit values in both normal and anemic animals. Tumorgrowth in anemic animals was slower than in normal animals, and rhEPOadministration did not influence tumor growth in either group. Tumorblood flow in anemic animals was lower than in control animals and wasonly increased in larger tumors in animals in which anemia was preventedby prophylactic rhEPO application. Tumor oxygenation, determined uslag polarographic needle electrodes and oxygen partial pressure histography, was poorer in anemic animals than in normal animals. Thisreduction could be reversed partially, but not compensated fully byrhEPO treatment in smaller tumors (@1.4mi). These changes suggest thatrhEPO, by Improving tumor oxygenation, may increase the efficacy ofstandard radiotherapy in anemic animals and may be of use in anemictumor patients in whom the success of radiotherapy or 02-dependentchemotherapy might be limited by tumor hypoxia.

INTRODUCTION

The aberrant microenvironment found in most experimental rodentand human malignancies can have a profound effect on tumor growthand response to various nonsurgical modalities (see, e.g., Refs. 1—3).For example, the presence of low 02 levels can confer cell protectionfrom sparsely ionizing radiation and some chemotherapeutic agents,and much evidence exists suggesting that the presence of hypoxia inhuman tumors may be a major reason for the failure of conventionalradiation therapy in the local control of malignancies (e.g., Refs. 4—6)and/or may be responsible for the development of an aggressivephenotype (7, 8). This problem may be exacerbated in situations inwhich the oxygen-carrying capacity of the blood is decreased. Onesuch situation occurring commonly in tumor patients is progressiveanemia (e.g., in approximately 50% of stage IV lung and prostatecancers3). Its etiology is multifactorial and not always associated with

acute or chronic bleeding. Many patients present with anemia beforethey receive cytotoxic therapy and even if their bone marrow is notinvaded by tumor cells (9). In these patients, factors such as deficiency of erythropoietic factors or suppression of erythropoietin,immune or nonimmune hemolysis, hemodilution, hypersplenism with

shorter RBC survival, bone marrow infiltration, nutritional causes

(iron, folate, vitamin@ 2 deficiencies), concurrent infections (hematophagocytosis), and paraneoplastic syndromes may play a role.

Numerous studies have investigated the relationship between anemia and response to radiotherapy (for a recent review, see Ref. 10)

Received5/21/96;accepted8/29/96.The costs of publication of this article were defrayed in part by the payment of page

charges. This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

I Supported by Stiftung Rheinland-Pfalz fuer Innovation (836-386261/49) and the

Deutsche Krebshilfe (Grant M 40/91 Va 1).2 To whom requests for reprints should be addressed. Phone: 49-6131-395929; Fax:

49-6131-395774.3 H. Ludwig, personal communication.

4728

Blood Flow, Oxygenation, and Bioenergetic Status of Tumors after ErythropoietinTreatment in Normal and Anemic Rats'

Debra K. Kelleher, Ulrike Matthiensen, Oliver Thews, and Peter Vaupel2

lnstitute of Physiology and Pathophysiology, Johannes Gutenberg-University of Mainz. Duesbergweg 6. D-55099 Mainz. Germany

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Table 1 RBC-related parameters in rhEPO and isotonic PBS-treated control and anemicanimalsnHematocrit

(v/v)RBCcount

(l06/pJ)cHb (glliter)Meancorpuscular volume

(fl)Meancorpuscular hemoglobin

(pg)Meancorpuscular cHb

(g/liter)Control

Control + rhEPO43 440.42±0.Ofl a

0.59±0.0116.5±0.1@ a

8.6±0.11141±@ a

184±2165.1±0.8@ a

69.4±0.7121.8±0.3

22.8± 1.2335±2@ a

311±21Anemic

Anemic + rhEPO28 260.30±0.Ofl a

0.48 ±0.0214.6±0.3@ a

7.0 ±0.3 195@ 6@ a

143 ±7167.3±1.8

69.7 ±1.320.5±0.3

20.4 ±0.4313±7 —@b

283 ±111

ERYTHROPOIETIN AND TUMOR OXYGENATION

ap <0.001.

bp < o.os.

rats have shown that there is no significant production of antibodies againstrhEPO over this treatment period.5

Tumors

Control Group. Solid tumors were induced on day 7 after commencementof rhEPO or isotonic PBS treatment by s.c. injection of DS sarcoma ascitescells (0.4 ml; approximately l0'@cells/s.d) into the hind foot dorsum (foradditional details, see Ref. 23). The tumors investigated appeared as flat,spherical segments. They replaced the subcutis and corium completely andinvariably reached the avascular epidermis. Tumor volumes (V) were calculated by an ellipsoid approximationusing three orthogonaldiameters (d):V = @7r/6X d1 x d2 x d3.

Anemia Group. Solid tumors were induced on the hind foot dorsum asdescribed above for control animals. In addition, approximately 108DS sarcoma cells were injected i.p. on day 9. This induced an anemia that resultsprimarily from the development of a hemorrhagic ascites. In the rhEPOtreatment group, the development of this anemia could be prevented prophylactically.

Measurements

Surgical Procedure and Measurement of Blood Parameters. Final investigations took place in all animals on day 14after commencement of rhEPOor isotonic PBS treatment. Rats were anesthetized with sodium pentobarbital(40 mg/kg i.p., Nembutal; Sanofi Ceva, Paris, France). Polyethylene catheterswere surgically placed into the thoracic aorta via the left common carotidartery and connected to a Statham pressure transducer (Type P 23 ID; Gould,Oxnard, CA) for MABP measurement and into the left external jugular vein(for subsequent anesthetic administration). Throughout all experiments, animals lay supine on a heated operating pad, and the rectal temperature wasmaintained at 37.5°C.Animals breathed room air spontaneously. At the end ofa 30-mm stabilization period, arterial microblood samples were taken, and thehematocrit (standard microcapillary method), cHb (cyanmethemoglobinmethod,kit 124729; BoehringerMannheim),and RBC count (Neubauerhemocytometer) were investigated. The RBC indices mean corpuscular volume,mean corpuscular hemoglobin, and mean corpuscular cHb could then be

calculated. The arterial blood gas status was assessed using a pH/blood gasanalyzer(type 178;Ciba Corning, Fernwald, Germany). The 502 of the arterialblood was obtained nomographically (24). Tumors then underwent one of thefollowing measurements:

TBF. TBF was estimated using the ‘33Xeclearance technique. The mdicator (0.1 ml of a solution of ‘33Xein saline, 185 MBq/ml; MedgenixDiagnostic, Ratingen, Germany) was applied as a bolus injection through thearterial catheter into the thoracic aorta. A Geiger-counting tube (covered witha 6 @.un-thickHostaphanmembrane)was placed over the tumorsuch thatnocompressionofthetumorsurfaceoccurred.Shieldingofthesurroundingtissuewas carriedout using flexible lead sheeting such that only the tumor wasexposed to the counting tube. The registration of the washout process wasperformed using a linear rate meter (FHT 1100; FAG Kugelfischer, Erlangen,Germany) connected to a chart recorder (LS 64; Linseis, SeIb, Germany). Withregard to the full-scale counting rate (I = 3000 cpm), the mean statistical errorof the rate meter was up to ±5%.Registration of the washout process wascarried out until the registered activity returned to the background level. Forevaluation of TBF, the clearance curves were transferred to single-logarithmicpaper, and the course of the curves was approximated by exponential functions. An index was then used that takes into account both the initial activity

for each exponential function as well as the corresponding half-time (for moredetails, see Ref. 25). Additionally, a tumor tissue/blood partition coefficient (A)was used, in which the actual hematocrit of blood in the animal undergoingmeasurement was taken into account (26). TBF was then expressed in ml . g. min'.

Tumor Oxygen Tension Distribution. P°2values were determinedusingpolarographic needle electrodes (recessed gold in glass electrode; shaft diameter, 300 @m;diameter of the 02-sensitive cathode, 12 @.tm)with stainless steelshafts (of the hypodermic needle type) and p02 histography (model KIMOC

6650; Eppendorf, Hamburg, Germany; for more details, see Ref. 27). Amidline incision was made in the skin covering the lower abdomen, and the

Ag/AgCl reference electrode was placed between the skin and the underlyingmusculature. Calibration was performed in saline solutions equilibrated with

either air or pure N2 immediately before and after tumor p02 measurements. Asmall incision was made into the skin overlying the tumor, and the 02-sensitive

electrode advanced to a depth of approximately 1 mm. The electrode was thenadvanced automatically through the tissue in preset net steps of 0.7 mm. Thesesteps consisted of a rapid forward step of I .0 mm, immediately followed by aretraction of 0.3 mm to minimize tissue compression artifacts. The 02 probewas removed automatically from the tissue at the end of the p02 measurement.Four or five parallel electrode tracks were evaluated in each tumor, and aminimum of 70 P°2 readings was obtained for each tumor. Using an online

computing system, data were pooled for individual tumors and for eachtreatment group, and P°2 frequency distributions were plotted with classwidths of 2.5 mm Hg (27).

Metabolite Concentrations. Tumors arid thigh adductor muscle were rapidly frozen in liquid N2,ground to a fine powder using a pestle and mortar, andsubsequently freeze-dried. Thereafter, glucose and lactate concentrations wereassayed enzymatically using standard test kits (kits 245158 and 256773;Boehringer Mannheim). For determination of AlP, ADP, and AMP, 2—3-mgaliquots of freeze-dried tissue were extracted with 0.3 Mperchloric acid andcentrifuged, and the supernatant was neutralized with 2 MKOH. The concentrations of the adenylate phosphates were then determined using reversedphase high-pressure liquid chromatography at 254 nm. The isocratic separationwas performed by a Supersphere Rp 18 end-capped column (250 X 4 mm;Knauer, Berlin, Germany) and a guard cartridge system (5 X 4 mm; Knauer).The mobile phase consisted of0.05 MNH4H2PO4,0.01 Mtetrabutylammoniumhydroxide, and 11.5%acetonitrile (v/v), adjusted to pH 6.4. The flow rate was0.9 mI/mm, and the sample size was 50 gd. Concentrations of all metabolitesare expressed as @moVgtissue wet weight (for a general description, see Ref.28).

Statistical Analysis

Results are expressed as means ±SE with the numbers of experimentsindicated in brackets. The significance of the differences between the variousgroups was assessed using the Wilcoxon test for unpaired samples. Resultswere considered as being significant if P values were less than 5% (P < 0.05).

RESULTS

cHb of the blood in anemic animals was found to be reducedsignificantly when compared with control animals (from 141 ±2 to95 ± 6 glliter; P < 0.001), thus confirming the induction of atumor-associated, normochromic, normocytic anemia in the expenmental system used (Table 1). Application of rhEPO caused significant increases in cHb in animals of the control (184 ± 2 g/l;P < 0.001) and anemia (143 ± 7 g/l; P < 0.001) groups, thusS Dr. W. Rebel, personal communication.

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Table 2MABP. arterialblood gases, pH. @°2'oxygencontentMABPP°2@0a50202

contentn(mmHg)(mm Hg)(mm Hg)pH(saturation %)(ml O@/mlblood)43

44141±3

143 ±476±1

75 ±242±1

44 ±I7.43±0.Ofl b

7.41 ±0.01 195±1

94 ±10.18±o.Ori c

0.23 ±0.01128

26l19±3@d 129±3173±2 75±138±l—@d

43 ±117.40±0.0ljd7.37±0.01193±1

94±10.l2±0.Oflc0.19±0.011

Tumor volume@ 1.4 mlTumor volume > 1.4mlControl0.85

±0.06 (16)―0.60 ±0.05(12)Control+ rhEPO0.73 ±0.06 (23)0.54 ±0.05(10)Anemic0.58

±0.06 (16)0.44 ±0.06(7)Anemic+ rhEPO

a Number of tumors i0.54

±0.07 (10)

s indicated in parentheses.0.61

±0.03(14)

ERYTItROPOIETIN AND TUMOR OXYGENATION

ControlControl+ rhEPOAnemicAnemic+ rhEPO

a pCO2, carbon dioxide partial pressure.

bp < 0.01.Cp <0.001.

dp < 0.05.

in control animals, although TBF still tended to decrease with increasing tumor volume. Upon rhEPO administration to the anemia group,this size dependency was no longer evident.

Oxygen availability to the tumor tissue (calculated as the product ofarterial blood 02 content and TBF) was higher in tumors of controlanimals than in untreated anemic animals (Fig. 2; P < 0.01 for bothgroups of tumors; lines connecting corresponding data points in Figs.2, 4, 5, and6 do not imply a linearrelationshipin betweentherespectiveparameters).rhEPOtreatmentofcontrol animalsresultedinno significant changes in oxygen availability. After rhEPO treatmentof the anemia group, the oxygen availability in the larger tumors wasno longer significantly different from that of control animals, whereas,in the smaller tumors, no significant improvement could be achieved.

After pooling of tumor P°2data for each treatment group, cumu

E0E0>

0E

time after implantation (days)

Fig. 1. Tumor volume as a function of time after tumor implantation. Volumes weremeasured in control (0), rhEPO-treated (Lx), and anemic (0) animals, and in animals inwhich anemia was prevented by prophylactic rhEPO administration (0). Each data pointindicates the mean value (bars, SE) for a minimum of 68 tumors.

Table3 TBFas measuredby the ‘33Xeclearancetechniquein rhEPOand isotonicPBS-treated control and anemic animals

TBF (ml â€g̃@'@

fulfilling the recommended treatment criteria for patients (increase incHb >20 g/liter, cHb >100 g/liter). The tumor-induced anemia couldtherefore be alleviated completely by the application of rhEPO.

The MABP and the arterial blood gas and pH status, together withthe 502 and 02 content of animals in the four experimental groups, isshown in Table 2. Although no significant changes were seen inarterial P°2' the MABP in anemic animals was significantly lowerthan in control animals (P < 0.001). This difference was reversedpartially after rhEPO administration to the anemia group. Additionally, rhEPO administration to animals of the anemia group resulted ina slight increase in arterial blood carbon dioxide partial pressure anda small decrease in arterial blood pH. Anemia caused a significantdecrease in arterial blood 02 content (P < 0.001). rhEPO administration to either control or anemic animals led to a significant increasein 02 content (P < 0.001), which tended to mirror those changes seenin cHb. In the case of the rhEPO-treated anemia group, the arterial 02content was then no longer significantly different from that of controlanimals.

Anemia in animals with hemorrhagic ascites was found to have asignificant effect on tumor growth. Seven days after implantation, the

mean volume of tumors in nonanemic animals was 1.25 ±0.05 ml(n 163), whereas in animals with tumor ascites (treated with rhEPOor untreated), the mean volume of tumors was 0.75 ± 0.04 ml(n 90; P < 0.001). For comparison, tumor volume growth in thedifferent treatment groups is shown in Fig. 1. The application ofrhEPO had no effect on tumor growth in either the control or anemia

group. Because in this tumor model a significant correlation has beenfound between both tumor volume and tissue oxygenation, and tumorvolume and blood flow (29), further investigations in this study werecarried out on tumors of comparable size ranges. Thus, in the control

group, tissue oxygenation, blood flow, and bioenergetic status measurements were carried out on day 7 after s.c. tumor implantation,when tumors had a mean volume of 1.25 ±0.05 ml (n 163), andin the anemia group on day 10, when tumors had a mean volume of1.39 ±0.08 ml (n = 94).

‘33Xeclearance was used to measure TBF, and results from theseinvestigations are shown in Table 3. As mentioned above, experiments were carried out at times when the mean tumor volumes for thefour experimental groups were comparable. Subsequently, for moredetailed analysis, tumors were divided into groups of two volumeranges (l.4 ml and >1.4 ml), such that volume-dependent phenomena could be identified, in addition to changes occurring due toanemia and/or application of rhEPO. As reported previously for thisexperimental tumor (29), a correlation between tumor volume andTBF was again found in control animals (r = —0.45),whereby TBFdecreased with increasing tumor volume. This tumor volume dependency was also evident after rhEPO administration to control animals.Application of rhEPO to control animals led to a significant reductionof TBF in the group of smaller tumors (P < 0.05). A similar trend,although not significant, was also seen in the larger tumors. When the

control and anemia groups were compared, TBF in the smaller tumorsof anemic animals was found to be significantly lower (P < 0.01) than

4730

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ERYTHROPOIE'FINANDTUMOROXYGENATION

volume range (P < 0.001), although again a recovery to values foundin control, nonanemic animals could not be achieved (Fig. 5, squares).In the case of the median P°2and the low p02 fraction, application of

16 rhEPO to control animals caused no significant changes (Fig. 5,triangles).

@,‘ No clear tumor volume-dependentrelationship was seen for the.@ 14 tissue concentrations of either glucose (1.66 ± 0.06 and 1.43 ± 0.07

,@ @.tmoVg, for small and large tumor volume groups, respectively) or

2@ AlP(0.73±0.06and0.81±0.03@mol/g,forsmallandlargetumor,g,i 2 volumegroups,respectively),andnosignificantchangeswereobi@' servedaftertheinductionof anemiaor theadministrationof rhEPO.:@ Inthecaseoftumorlactatelevels,however,adistinctincreasewas.@ 10 found with increasing tumor volume in all treatment groups (Fig. 6),

@ with much higher levels of lactate being found in the larger tumors of@ 8 theuntreatedanemiagroup(P < 0.05).

DISCUSSION

6 For cancer patients, progressive anemia is a common clinical prob1cm and may be at least partially responsible for poor response to

_________________________________ radiotherapyin somepatients,becauseseveralstudieshavesuggested‘to@ 0.5@ I :@ ‘@ :5@ 2:0@ 2.5 thatlow hemoglobinlevelsareanindicatorof poorprognosisin

patientstreatedwith radiotherapy(for a recent review,see Ref. 10).Although rhEPO has been shown to be effective and safe for the

Fig. 2. Tumor oxygen availability in control (0), rhEPO-treated (Li), and anemic ( 0 ) correction of malignancy-associated anemia in patients (15—22), noanimals, and in animals in which anemia was prevented by prophylactic rhEPO admin- . . .istration (0), as a function of tumor volume. Each data point indicates the mean value clinical studies are available that show whether rhEPO therapy can(bars, SE)for a minimum ofseven tumors. Lines connecting corresponding data points donot imply a linear relationship between the respective parameters.

lative frequency distributions of P02 values were constructed asshown in Fig. 3. Anemia resulted in a drastic left shift of the P°2distribution, indicating a shift to lower PO2 values and a substantialdeterioration of tumor oxygenation (Fig. 3, diamonds). In smallertumors, this deterioration could be alleviated slightly by the application of rhEPO (Fig. 3, squares). Full recovery to levels found incontrol animals could not be achieved by this maneuver (Fig. 3,

circles). The effect of anemia and rhEPO application in the anemiagroup on the cumulative frequency distribution was most pronouncedin the smaller tumors (l.4 ml; Fig. 3, top). In the larger tumors, thesedifferences became less distinct (>1.4 ml; Fig. 3, bottom). Administration of rhEPO to normal animals caused no major changes in theform of the cumulative frequency curve, irrespective of tumor volume.

Comparable effects on tumor tissue oxygenation were again seenwhen median P°2values were considered (Fig. 4). In control animals,a tumor volume dependence of this parameter was seen, with themedian P°2decreasing with increasing volume (r = —0.377;Fig. 4,circles). The median P°2was decreased additionally in animals withuntreated anemia (Fig. 4, diamonds). This reduction could be alleviated partially by rhEPO treatment in the group of small tumors (Fig.4, squares; P < 0.001 in both cases). However, the median p02values were still significantly lower than those found in controlanimals (P < 0.001), indicating that a return to preanemia levels wasnot possible with the rhEPO treatment protocol used. Likewise, whenthe fraction of P°2measurements between 0 and 2.5 mm Hg wasconsidered (indicating less than half-maximum radiosensitivity), asimilar trend was seen (Fig. 5). In control animals, the size of thisfraction increased with increasing tumor volume (Fig. 5, circles). Inanemic animals, this size dependency is no longer evident (Fig. 5,dianwnds), although with both tumor volume ranges, a significantly Fig. 3. Cumulative frequency distributions of oxygen partial pressures measured inlarger low p0 fraction is seen than that measured in control tumors tumorsof twovolumeranges(top, l.4 ml;bonom,>1.4 ml)in control(0), rhEPO

. 2 . . . . treated (ti), and anemic ( 0 ) animals, and in animals in which anemia was prevented by

(P < 0.01 in both cases). The administration of rhEPO to the anemia prophylacticrhEPOadministration(0). Eachcurveindicatetvaluesobtainedfromagroup resulted in a decrease in this fraction for tumors of the smallest minimumof fivetumorsand 573individualp02 measurements.

4731

tumor volume (ml)

5

oxygen partial pressure (mmHg)

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0 0.5 1.0 1.5 2.0 2.5

ERYTHROPOIETINAND TUMOR OXYGENATION

likelihood of distant metastases (32). Reports of experimental studies

are also scarce. In one study by Joiner et a!. (33), the effect of rhEPOon radiosensitivity in a mouse tumor model was investigated. Here,rhEPO was found to have no influence on tumor radiosensitivity,although it should be noted that at the time of radiotherapy, only amild anemic state (hematocrit: @0.39)had been induced.

In the present study, the animal model used allowed the inductionof a moderate tumor-associated anemia (hematocrit: @0.29,equivalent to a clinically relevant anemia found in many tumor patients) atthe time of tumor oxygenation and blood flow measurements. rhEPOat a dose of 1000 lU/kg was shown to cause significant increases inthe hematocrit values and in hemoglobin content both in control andanemia group animals bearing DS sarcomas, with a correction topreanemia levels in the latter group.

When tumor growth was investigated, tumor-associated anemiacaused a significant slowing of tumor growth, a phenomenon that hasalso been reported for C3H tumors in mice (34). This correlates withthe findings of Tannock and Steel (35), who found a slowing down oftumor growth in animals exposed to respiratory hypoxia. In thepresent study, however, the tumor-induced anemia cannot be responsible for the inhibition of solid tumor growth, because this inhibitionalso occurred in animals in which anemia was prevented by prophylactic rhEPO administration. In an animal model, Hartveit and Halleraker (36) have shown that the growth of a solid tumor can beinhibited in the presence of an ascites tumor of the same cell line. Inthe present study, the malignant ascites may therefore be exerting a

similar inhibitory effect. That rhEPO treatment was found to have nosignificanteffecton tumorgrowtheitherin controlor anemicanimalsis a significant finding with respect to the use of rhEPO in the clinicalsetting. Similar findings have been made in vitro, where rhEPO wasfound to have no stimulating effects on the growth of human tumorcell lines (37—39),although erythropoietin receptors are expressed in

10

5

2

1

0.50

a)IEE

di0.CCO@00E

I-

L0.5 1.0 1.5 2.0 2.5

tumor volume (ml)

Fig. 4. Median pO@values of tumors in control (0), rhEPO-treated (Lx),and anemic(0 ) animals,andin animalsin whichanemiawas preventedby prophylacticrhEPOadministration (El)' as a function of tumor volume. Each data point indicates mean values(bars, SE) for a minimum of five tumors.**, P < 0.01. Lines connectingcorrespondingdatapointsdo not implya linearrelationshipbetweenthe respectiveparameters.

improve tumor oxygenation, a parameter that is known to modulatethe effects of sparsely ionizing radiation and that has been shown tobe a significant and independent oncological parameter for predictionof patient survival and local recurrence (7, 30, 3 1) and for the

4732

ô)00E

C0

CO

C00C000tO

0(0

Fig. 6. Lactate concentrations in tumors in control (0), rhEPO-treated (Lx),and anemic(0 ) animals,andin animalsin whichanemiawas preventedby prophylacticrhEPOadministration (0). as a function of tumor volume. Each data point indicates the meanvalue (bars, SE) for a minimum of seven tumors. Lines connecting corresponding datapoints do not imply a linear relationship between the respective parameters.

0)IEEto

0

tumor volume (ml)

Fig. 5. Fraction of measured p02 values between 0 and 2.5 mm Hg (indicating less than

half-maximal radiosensitivity) in tumors in control (0), thEPO-treated (is), and anemic(0 ) animals,andin animalsin whichanemiawas preventedby prophylacticrhEPOadministration (0), as a function of tumor volume. Each data point indicates the meanvalue (bars, SE) for a minimum of five tumors. **, P < 0.01. Lines connectingcorresponding data points do not imply a linear relationship between the respectiveparameters.

2.5

tumor volume (ml)

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ERYTHROPOIETINAND TUMOR OXYGENATION

some of the cell lines investigated.6 In an in viva study, rhEPO wasfound to cause a small slowing of growth rate in mouse tumors athigher rhEPO doses, although growth of the same tumor line in vitrowas not affected by the presence of rhEPO (33).

Anemia resulted in a decrease in the 02 content of arterial bloodthat could be restored to control values by rhEPO application. Whentumor oxygen availability was calculated, a decreased 02 availabilitywas seen in tumors in anemic animals as compared to control animals.Anemia resulted additionally in a decrease in the MABP, which was

restored partially to control values after rhEPO administration.When the results of tumor oxygenation measurements were con

sidered, anemia led to a deterioration of tumor tissue oxygenation(seen as a shift to lower P°2values, a decrease in the median P°2'andan increase in the fraction of low p02 readings) despite an improvedrelease of oxygen to the tissue from RBCs in anemic animals [resulting from a right shift of the oxyhemoglobin dissociation due to anemia(34), a possible increase in 2,3-diphosphoglycerate in RBCs (40, 41),and a slight arterial acidosis]. This is in agreement with the findingsof McCormack et al. (34), who carried out radiotherapy studies, theresults of which suggested that tumors grown in anemic mice are more

radioresistant and have a higher hypoxic fraction than those grown in

control mice. In a study by Rojas et a!. (42), however, anemia(induced by bilateral kidney irradiation given several months beforetumor implantation) produced an increase in radiosensitivity thatcould be enhanced further by RBC replacement, whereas Joiner et a!.(33) found no effect of hematocrit on tumor radiosensitivity. In bothof these studies, a possible role of physiological adaptation to hematocrit changes occurring over prolonged periods of time was discussed. In the present study and that of McCormack et a!. (34), anemiawas only evident for a period of days, although a deterioration oftumor oxygenation and decreased tumor radiosensitivity were nevertheless seen. Reasons for these conflicting findings are at present notapparent.

When anemia was corrected by application of rhEPO in the presentstudy, the deterioration of tissue oxygenation could only partially bereversed in terms of a right shift in the cumulative frequency distribution curves, an increase in median p02 values, and a decrease in thehypoxic fraction. Because no increase in TBF was seen in animals ofthe anemia group in the present study after rhEPO administration, theincreased tumor oxygenation can presumably be attributed to anincrease in the 02 content of the arterial blood. Alterations in TBFafter rhEPO were only seen in control animals, whereas in the groupwith tumor volume 1.4 ml, a significant reduction in TBF was seen.This is most likely due to increases in viscous resistance to flow in thetortuous, chaotic tumor vasculature resulting from increased bloodviscosity as a consequence of the higher hematocrit, which developsafter rhEPO application. Sevick and Jam (43), in a study on the effectof hematocrit on intratumor blood viscosity, showed that blood viscosity increases exponentially as a function of hematocrit, with the“threshold―section leading into the steeper part of the curve occurringat a hematocrit of @0.45.For even small increases in hematocrit>0.45, pronounced increases in blood viscosity occur, and therefore,after rhEPO administration to control animals in this study, in whicha hematocrit of 0.59 was obtained, a large increase in viscosity wouldbe expected. Even so, the increased arterial blood 02 content, whichoccurs as a result of the increased hemoglobin content of the blood incontrol animals receiving rhEPO, is sufficient to compensate for the

reduced oxygen delivery occurring as a result of the increased resistance to flow (decreased flow), such that the oxygen availability to thetumor tissue is not compromised. This is also seen when tumor

oxygenation is measured, and no changes in the form of the cumulative frequency curve or median p02 occur for tumors in controlanimals receiving rhEPO.

When the metabolic parameters measured are considered, the increased lactate levels found with increasing tumor volume, in particular in the larger tumors in untreated anemic animals, suggest ashifting of energy metabolism to glycolysis. In addition, the lack of

decreases in tumor ATP levels in this experimental model of anemiasuggests that oxygen is not a limiting factor in energy metabolism and

that 02 per se cannot be the principal parameter for the genesis oftumor necrosis, as long as glucose is adequately available, a findingthat is in agreement with that of Gerweck et a!. (44).

In conclusion, the changes in tumor oxygenation seen in animals ofthe anemia group with smaller tumors after rhEPO treatment suggestthat rhEPO administration might be of use in “sensitizing―sometumors to sparsely ionizing radiation and oxygen-dependent chemotherapy in anemic animals, and that this form of anemia correctionmay be effective in increasing tumor radiosensitivity in some anemictumor patients in whom the success of radiotherapy might be limitedby tumor hypoxia. Additional studies of both an experimental and aclinical nature may be warranted to further elucidate the limitations ofthe effectiveness of rhEPO in increasing tumor radiosensitivity, especially in terms of tumor mass:body weight ratio.

ACKNOWLEDGMENTS

The rhEPO used in this study was donated kindly by Boehringer Mannheim(Mannheim, Germany). DS sarcoma was provided by Dr. H. Loehrke from the

Tumor Bank of the German Cancer Research Center in Heidelberg. Theauthors are indebted to Simone Dahms-Praetorius and Anise Kosan for theirskilled technical assistance in carrying out this study.

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1996;56:4728-4734. Cancer Res   Debra K. Kelleher, Ulrike Matthiensen, Oliver Thews, et al.   after Erythropoietin Treatment in Normal and Anemic RatsBlood Flow, Oxygenation, and Bioenergetic Status of Tumors

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