glucose uptake, lactate release, ketone body turnover, … · determination of glucose, lactate,...

(CANCER RESEARCH 48, 7264-7272. December IS, 1988]

Glucose Uptake, Lactate Release, Ketone Body Turnover, Metabolic Micromilieu,and pH Distributions in Human Breast Cancer Xenografts in Nude Rats1

F. Kallinowski,2 P. Vaupel, S. Runkel, G. Berg, H. P. Fortmeyer, K. H. Baessler, K. Wagner,

W. Mueller-Klieser, and S. Walenta

Department of Radiation Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114 [F. K., P. VJ; Department of AppliedPhysiology Â¡S.R., G. B., W. M-K., S. W.] and Department of Physiological Chemistry [K. H. B., K. W.], University of Mainz, D-6500 Mainz, and Department of AnimalExperimentation, University Clinics of Frankfurt, D-6000 Frankfurt/M. [H. P. F.Â¡,FRG

ABSTRACT

Glucose uptake, lactate release, ketone body utilization, spatial distribution of glucose, lactate, and ATP concentrations as well as tissue pHdistributions were systematically investigated in s.c. and/or "tissue-isolated" human breast cancer xenografts in T-cell-deficient rnu/rnu rats.

Large variations in all parameters were detected within and betweentumors indicating a very nonuniform substrate turnover. Glucose wastaken up by all xenografts. Glucose consumption rates increased withincreasing glucose availabilities, implying that the glucose uptake ismainly determined by the efficiency of nutritive tumor blood flow. Theaverage glucose uptake was 0.37 Â¿imol/g/minin medullary and 0.26 nnw\jg/min in squamous cell carcinomas of the breast. At wet weights below Sg, medullary breast cancers consumed more glucose than squamous cellcarcinomas (IT < 0.05). Most tumors (97%) released lactate in an amountlinearly related to glucose consumption. The lactate production of medullary (0.33 Â¿iniÂ»!e min) and squamous cell (0.31 /imol/g/min) breastcancers was similar. In general, the xenografts utilized ketone bodies, ÃŸ-Hydroxybutyrate was consumed by 82% and acetoacetate by 73% of thetumors, the uptake rates being linearly related to the respective availabilities. The mean uptake of /J-hydroxybutyrate was 3.48 nmol/g/min andthat of acetoacetate 2.56 nmol/g/min. No significant differences wereseen between medullary and squamous cell breast cancers. The /9-hy-droxybutyrate/acetoacetate ratio in the tumorvenous blood rose withdecreasing tumor blood flow indicating the development of hypoxia atadvanced growth stages. Glucose, lactate, and ATP levels were all veryheterogeneously distributed in medullary and squamous cell tumors ascompared with normal tissue. No relationship was evident between thespatial distribution of concentrations of these three substrates. Thexenografts were acidotic compared with pH values in normal subcutis.The mean tissue pH in medullary breast cancers was 6.81 Â±0.25 (SD).Compared with these values, the tissue pH distribution in squamous cellbreast cancers was shifted to significantly higher values. The mean pHof the latter tumors was 7.04 Â±0.19(21' < 0.001 ). From the experimental

data presented there is clear indication that the metabolism of thexenografts investigated was mainly determined by the efficiency of nutritive blood flow, i.e., by substrate availability, and not by the metabolicdemand of the cancer cells.

INTRODUCTION

Based on in vitro experiments, Warburg (1) postulated a highaerobic glycolysis to be the outstanding biochemical characteristic of neoplastic tissue. The investigation of this hypothesisunder in vivo conditions was permitted by the development of"tissue-isolated" tumor preparations using kidney, ovary, testis,

or an inguinal fat pedicle as implantation sites (2-8). In thesestudies, blood flow, oxygen consumption, glucose uptake, andlactate production of malignant rat tumors were measured.Recently, the inguinal implantation site was used to study theturnover of further substrates like ketone bodies and amino

Received 6/3/88; revised 8/9/88: accepted 9/2/88.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 inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1Supported by Grants Va 57/2-4 and Mu 576/2-3 from the Deutsche For

schungsgemeinschaft.1To whom all requests for reprints should be addressed, at Department of

Radiation Medicine. Massachusetts General Hospital. Boston, MA 02114.

acids (9-12). These in vivo studies revealed that a particularmetabolic micromilieu develops in many malignant rodent tumors due to severe restrictions of convective and diffusivetransport. This metabolic micromilieu is characterized by hypoxia and even anoxia, a general deprivation of nutrients andan insufficient removal of metabolic waste products, predominantly lactic acid, causing tissue acidosis (13). For humantumors in vivo, very few direct determinations of substrateturnover are available (14, 15). Since the available data wereobtained from tumors with widely varying histologies, no correlation between metabolic micromilieu and treatment responseis possible. Furthermore, systematic investigation of patients'

tumors, i.e., measurements on tumors from one cell line atdifferent growth stages, cannot be performed due to the needof immediate therapeutic intervention. Therefore, the xeno-transplantation of human tumors as "'tissue-isolated'" prepara

tions into nude rats was performed in order to gain furtherinsight into the nutrient uptake as a potential modulator oftumor growth and cancer-related cachexia. As a first approach,breast cancers were investigated due to their frequency andmultimodal treatment. Regional distributions of important substrates and metabolites (glucose, lactate, and ATP) were determined using s.c. xenotransplants of the same tumor lines.Furthermore, tissue pH distributions were measured in thesetumors. Knowledge of tumor pH is important since tissueacidosis can influence the proliferation of malignant and normalcells (16), the substrate utilization (17), the metastatic ability(18), and the efficacy of radiotherapy, chemotherapy and hy-perthermia (19, 20). The study reported here provides for thefirst time detailed data on in vivo metabolism and regionalmetabolic micromilieu of human tumor tissue. It is concludedthat this approach may help in evaluating therapeutically relevant parameters to be determined in human tumors in patients.

MATERIALS AND METHODS

Animals and Tumors. Homozygous, athymic nude rats (WAG/Fra-rnu/rnu) were obtained from a colony established at the Department ofAnimal Experimentation, University Clinics, Frankfurt (FRG). Breeding and maintenance of these rats have been described previously (21,22). In brief, the rats were maintained under defined environmentalconditions (temperature, 25 Â±1Â°C;relative humidity >70%; 12-h light-

dark cycles). The rats were supplied with a highly digestible, energy-and protein-rich diet (Altromin 1414, Altromin, Lage/Lippe, FRG)and drinking water (pH 2.5) ad libitum. At the day of investigation,food was removed at 8:00 a.m. The experiments began 2-4 h later.

Tumor tissue was obtained from pre- and postmenopausal womenduring surgery for breast cancer at the Department of Gynaecology andObstetrics, University Clinics of Frankfurt (FRG). After histolÃ³gica!confirmation of the diagnosis, the tumor tissue was directly transplanted to nude (nu/nu) mice and serially passaged thereafter. Fromthis "tumor bank," xenografts in the fourth to 93rd generation were

grafted into nude rats. Six medullary and two squamous cell carcinomalines were investigated. Glucose uptake, lactate release, and ketonebody utilization were determined using "tissue-isolated" preparations

7264

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METABOLISM OF HUMAN BREAST CANCER XENOGRAFTS IN VÃŒVO

as previously described (21, 23). Measurements of blood flow, oxygenconsumption, and tissue oxygÃ©nationas well as histolÃ³gica! studieswere performed as reported by Vaupel et al. (21). Spatial distributionsof glucose, lactate, and ATP concentrations as well as tissue pHdistributions were measured in s.c. flank tumors (21). The tumors wereused at average wet weights of 2-3 g (4 to 6 weeks after transplantation).At that time, the rats were up to 110 days old (mean body weight Â±SD, 280 Â±44 g).

Experimental Protocol for the Investigation of "Tissue-isolated" Tu

mors. During pentobarbital-Na anesthesia (Nembutal, Ceva, Paris; 35-40 mg/kg i.p.) catheters were placed into the external jugular vein, thecommon carotid artery, and the tumor vein (former inferior superficialepigastric vein) using an operating microscope when necessary (Olympus MTX; Olympus Optical Co. Ltd., Tokyo, Japan). The mean arterialblood pressure was continuously monitored via the arterial cannula(Statham pressure transducer, type P23 ID, Gould blood pressuremonitor, type SP 1400, Gould, Oxnard, California). Arterial and tumor-venous blood samples were taken repeatedly under steady stateconditions. Any blood loss due to sampling was adequately replenishedby fresh donor blood via the catheter in the jugular vein. Fresh donorblood was derived from nude rats of the same litter, which weremaintained under identical conditions and implanted with tissue of thesame cell lines. Arterial blood was taken from the carotid artery ofheparinized animals (350 USP units/kg) about l h before use, storedon ice (7"= 4Â°C)and warmed to body temperature for infusion.

Total blood flow through the tumors was directly determined bytimed collection of blood draining from the tumor vein. Thereby,outflow of blood from the tumor-venous cannula per unit time wascollected in vials with known weight (3810; Eppendorf, Hamburg,FRG). Volume flow was calculated taking into account the weight ofthe collected blood, a density of 1.06 g/ml and the wet weight of thetumor excised after the completion of the experiment. During thecollection period, resistance to flow within the catheter did not exceed5 mmHg-min/ml.

Throughout the experiments, the animals were placed on a thermo-statized heating pad in order to keep the rectal temperature at 37 Â±1Â°C.At the end of the experiments, the tumors were excised, weighed,

and examined by standard histolÃ³gica!techniques.Determination of Glucose, Lactate, and Ketone Body Turnover. The

arterial and tumor-venous blood samples were deproteinated immediately with ice-chilled perchloric acid (glucose, 0.33 M;lactate and ketonebodies, 1 M). The substrate concentrations were determined enzymati-cally. For measurements of blood glucose levels, a hexokinase/G6P-DH-method was used (gluco-quant; Boehringer Mannheim, Mannheim, FRG). The lactate concentrations were determined by an enzymatic test according to Hohorst (24). The ketone bodies were measuredwith enzymatic micromethods (25). From the actual TBF3 and the

substrate concentrations, the following parameters (per unit weight)were calculated (26):

Availability (or supply) = arterial concentration x TBF (A)

Consumption (or uptake) or release rate= arterial-tumor-venous concentration difference x TBF (B)

arterial-tumor-venous concentration differenceUtilization = â€”¿� ; (C)

arterial concentration

Assessment of the Spatial Distribution of Glucose, Lactate, and ATPin s.c. Xenografts. The metabolic micromilieu was assessed in s.c.xenografts of human breast cancers by metabolic imaging using abioluminescence technique (27, 28) modified for the use in tumor tissueby Mueller-Klieser et al. (29). Following this protocol, cryobiopsieswere taken from normal and cancerous tissues with special tongsprecooled in liquid nitrogen (30). Then, the frozen tissue samples wereembedded in tissue tek (Miles Laboratories Inc., Elkhart, IL) and wereserially sectioned at -25Â°Cin a cryomicrotome (type TE, SiÃ©e,Mainz,

FRG). With the exception of sections for glucose measurements, enzymes in the tissue slices were heat-inactivated after freeze-drying.

3The abbreviation used is: TBF, tumor blood flow.

Concomitantly, an enzyme cocktail was prepared, containing all enzymes and cofactors necessary to link the substrates of interest to theluciferase luminescence reaction via NAD(P)/NAD(P)H redox systems.This solution was rapidly frozen and sectioned at -25Â°C as well. A

sandwich, formed from frozen sections of the tissue and of the enzymemixture, was placed upon a photographic film under a cover glass.Upon thawing, the enzymes diffusing into the tissue sections initiatedluminescence. In complete darkness, the photon emission is used forfilm exposure. After conventional processing, the films were evaluatedby microdensitometry and specific image analysis. Thus, regional distributions of glucose, lactate, and ATP have been obtained on a relativescale in serial sections, i.e., at adjacent locations, from human breastcancer xenografts.

Measurements of Tissue pH Distributions in s.c. Xenografts. TissuepH measurements were performed with steel-sheathed, miniaturizedneedle glass pH electrodes (type MI 408 B; Microelectrodes Inc.,Londonderry, NH). The performance characteristics of these electrodesboth in buffer solutions and tissues as well as the setup used have beendescribed previously (31). In the experiments reported here, the pHelectrode was inserted into the tumor tissue to an initial depth of 0.25-0.50 mm after careful removal of the overlying skin and suban is. Thereference electrode (macro calomel electrode; type 303, Ingold, Frankfurt/M., FRG) was routinely placed into a s.c. pocket approx. 4 cmaway from the insertion site of the pH electrode in order to minimizeany possible influence of different biopotentials of the tissues. Theinsertion sites were moisturized with 0.9% NaCl-solution (T = 34Â°C).

After obtaining stable readings (equilibration time, 10-15 min), the pHelectrodes were advanced in steps of 0.5 mm. 60-80 values weremeasured in each tumor (two to four electrode tracks). For controlvalues, pH measurements were performed in s.c. tissue at the lowerabdominal wall of nude rats. Here, the pH electrodes were insertedthrough a skin incision and progressively pushed forward at stepintervals of 1-2 mm. Three to five values were taken per animal.

Arterial blood samples were repeatedly collected from these animalsfor determination of respiratory gas and acid-base status (O2 and CÃ›2partial pressures and pH values; blood gas analyzer, type MT 33,Eschweiler, Kiel, FRG). The arterial hematocrit was measured bycapillary tube centrifugation. The oxyhemoglobin saturation of thearterial blood was obtained nomographically according to Bork el al.(32). Here again, any blood loss due to sampling was adequatelycompensated with fresh donor blood.

Statistical Evaluation. For the data obtained, descriptive statisticalparameters were calculated. Differences within and between groups oftumors were statistically evaluated using the Kruskal-Wallis H test andthe Mann-Whitney U test. Means Â±SE are given if not indicatedotherwise. Linear regression lines were calculated using a commerciallyavailable least square fit program (Statworks; Cricket Software, Philadelphia). Hyperbolic curves were fitted using a nonlinear maximumlikelihood least-squares estimation technique (software written by Dr.P. Okunieff, Department of Radiation Medicine, Massachusetts General Hospital, Harvard Medical School, Boston).

The pH data obtained were grouped in relative frequency histogramsin order to get an insight into the distribution of pH values within thetissues. For statistical tests, pH data measured in a single animal ortumor were represented by their median pH value.

RESULTS

Glucose Uptake, Lactate Release, and Ketone Body Utilizationof "Tissue-isolated" Human Breast Cancer Xenografts. A totalof 67 tissue-isolated preparations of human breast cancer xenografts in nude rats were investigated. 43 tumors out of 67xenografts were medullary mammary carcinomas and 24 tumors were squamous cell carcinomas of the breast. Relevantparameters related to the tumors investigated are compiled inTable 1. In both tumor types, glucose consumption rates werelinearly related to glucose availabilities (Fig. 1). At relativelyconstant arterial glucose concentrations the glucose supply wasmainly determined by nutritive blood flow. Blood flow through

7265



METABOLISM OF HUMAN BREAST CANCER XENOGRAFTS IN VIVO

Table I Glucose, lÃ¡claleand kelone body concentrations in the arterial andtumor-venous blood, arterio-lumor-venous concentration differences, arterialavailability and consumption or release rates measured in "tissue-isolated"

human breast cancers xenografted into nude ratsTotal TBF and oxygen consumption per unit tumor mass (I,,,) of these tumors

are given for comparison (21). Values are means Â±SE.

Breast cancer xenografts

15

Number oftumorsAnimalage(days)Body

weight(g)Tumorgrowth period(days)Tumorwet Â»eight(g)TBF

(ml/g/min)>'o: (dlOi/g/min)Arterial

[gl|Â°(m.M)Tumor-venous[gl|(mM)avD,,

(mM)Glucosesupply(pmol/g/min)gl

consumption(^mol/g/min)Arterial[la|(mM)Tumor-venous

[la](mM)â€¢,;>!>,(mM)LÃ¡clate

supply(*imol/g/min)LÃ¡claterelease(Â»jmol/g/min)Arterialjacac] (I-MITumor-venous

[acac](^M)avDKK(>iM)acac

supply(nmol/g/min)acacconsumption(nmol/g/min)Arterial

[fi-hb](Â¿IM)Tumor-venous[rf-bh](MM)avDa-hb

(MM)/i-hbsupply(nmol/g/min).-'lihuptake (nmol/g/min)Medullary4397

Â±1287Â±734

Â±12.35Â±0.280.17

Â±0.0210.4Â±1.27.86Â±0.365.24Â±0.222.63Â±0.221.25

Â±0.140.37Â±0.044.98Â±0.297.41

Â±0.372.43Â±0.240.79Â±0.090.31

Â±0.04lÃ®tÂ±6100

Â±418Â±719

Â±23Â±161Â±442Â±519Â±410Â±

14Â±1Squamous

cell2496

Â±2264Â±829

Â±12.23Â±0.270.10

Â±0.027.7Â±1.38.28Â±0.455.25

Â±0.253.02Â±0.340.85Â±0.140.26Â±0.035.43Â±0.529.12+0.663.69Â±0.420.43Â±0.060.30Â±0.04129

Â±9108Â±821

Â±1113Â±22Â±

185Â±1541

Â±644Â±159Â±35Â±3

* Abbreviations: gl. glucose; [ ]. concentration; avD. arterio-tumor-venous concentration difference; la. lÃ¡clate;vaD, tumor-venous-arterial concentration difference; acac. acetoacetate; iJ-hb, fi-hydroxybutyrate.

125

025

05 10 1.5 20 25 30 35 4.0

Fig. 1. Glucose consumption rates (KBi)per unit weight of medullary andsquamous cell breast cancer xenografts as a function of glucose availability(medullary tumors, â€¢¿�.y = 0.232* + 0.082, Ã•P< 0.001 ; squamous cell carcinomas,O,y= 0.187* + 0.106. 2P< 0.001).

both types of breast cancers investigated was compromisedalready in small tumors as evidenced by a flow-limited oxygenconsumption and large tissue areas with hypoxia and anoxia(21). Therefore, the glucose uptake of both medullary andsquamous cell breast cancers investigated was primarily limitedby the delivery of this substrate and not by the metabolicdemand of the cancer cell lines (Fig. 2). Since the tissue oxygÃ©nation,the oxygen consumption and the glucose uptake weregoverned by nutritive blood flow (21), linear relationships between the oxygen consumption, and the glucose uptake werefound (Fig. 3). The medullary carcinomas had higher bloodflow and glucose supply rates than squamous cell carcinomasand had correspondingly higher rates of glucose uptake atcomparable tumor sizes (Fig. 4, 0.41 versus 0.26 Â¿imol/g/min

10-

0.5

Vgilpmol/g/min)

0 01 02 0.3 0.4 05 0.6

TBFIml/g/minlFig. 2. Influence of tumor blood flow (TBF) on the glucose consumption ( I',, )

per unit weight of xenografted medullary and squamous cell human breast cancers(medullary, â€¢¿�,>â€¢=1.261x + 0.163, 2P< 0.001; squamous cell, O, >â€¢=1.391x +0.122, 2P< 0.001).

02 06 0.8 1.0 1.2 1.4

V., (iimol/g/min)

Fig. 3. Oxygen consumption (F02) per unit weight of medullary (â€¢)andsquamous cell (O) human breast cancer xenografts at various glucose uptake rates( y#). The lines indicate the respective trend (medullary, >â€¢=16.281*+ 4.295. IP< 0.001 ; squamous cell, y = 22.463* + 1.833, 2P < 0.01 ).

150

1.00-

0.50-

Vgilpmol/g/min)

02468

tumor wet weight (g)

Fig. 4. Relationship between glucose consumption ( l â€ž¿�,i per unit weight andtumor size of xenografted medullary and squamous cell breast cancers. The linesindicate the respective trend (medullary, â€¢¿�,y = -0.371 log AT-t-0.463, 2P < 0.01 ;squamous cell. O, y = -0.118 log x + 0.287, NS).

7266




for tumors <5 g; IP < 0.05). The glucose consumption per unittumor mass exhibited considerable intertumor variability inboth medullary and squamous cell carcinomas which is comparable to previously measured intertumor blood flow variability (21). The glucose uptake of the medullary breast cancersdecreased from 1.02 to 0.03 jumol/g/min with tumor sizesincreasing from 0.4 to 7.1 g. The respective consumption ratesof squamous cell cancers diminished from 0.66 to 0.02 /Â¿mol/g/min as tumor sizes enlarged from 0.2 to 5.5 g. During tumorpassage of the blood (and neglecting the impact of possibleshunt flow), about one third of the glucose available was extracted from both medullary and squamous cell breast cancers,the glucose utilization being comparable in both tumor types(medullary, 0.32 Â±0.02; squamous cell, 0.35 Â±0.03). Theglucose extraction was found to be independent of tumor wetweight, tumor blood flow or oxygen consumption.

All but two tumors released lÃ¡clate,the release rate of medullary tumors being similar to that of squamous cell carcinomas.For glucose consumption rates less than 0.75 ^mol/g/min thelÃ¡claterelease was linearly related to the glucose uptake (Fig.5). Two xenografts with consumption rales above Ihis valueshowed a nel lÃ¡claleuplake. No significant relalionship belweenthe lÃ¡clate turnover and the tumor wet weight or Ihe arteriallÃ¡claleconcentration was delecled. The glycolylic rale can bedelermined as Ihe ratio of the lÃ¡clalerelease and Ihe glucoseuplake assuming (a) steady slale condilions and (/>)glucose lobe the only substrale source of lÃ¡clale.Wilh Ihese assumplions,il was found lhat in the smaller tumors (approximately 1 g)50% of Ihe glucose taken up was released as lÃ¡clale.This ratioincreased with tumor growlh. In large breast cancer xenografts(lumor wel weighls, 5-8 g), an average of Iwo moles of lÃ¡clalewas produced for every mole of glucose laken up, yielding amean glycolylic rale of 100%. However, 10 of Ihe 67 xenograftsreleased more lÃ¡clatethan could be accounled for by glycolysisalone, indicaling lÃ¡clateproduclion from subslrales olher thanglucose (e.g., amino acids).

Ketone Bodies. 0-Hydroxybutyrale and aceloacetale were pre-dominanlly consumed by bolh medullary and squamous cellbreasl cancer xenografts, Ihe uplake being linearly relaled loIhe availabilily of Ihe respective substrale (Fig. 6). From Iheseresulls il has lo be concluded lhat, similar lo oxygen andglucose, Ihe uplake of ketone bodies is mainly determined by

1.0

0.8-

0.6

0.4-

0.2-

Vmlpmol/g/min)

Vgi(pmol/g/min)

0 0.25 0.50 0.75Fig. 5. LÃ¡claterelease ( K.) per unit weight of human breast cancer xenografts

as a function of the glucose uptake rate (medullary, â€¢¿�,y = 0.668* + 0.138, 2P <0.001; squamous cell. O, y = 0.902* + 0.077, 2P< 0.001).

20-

15-

10-

5-

uptake (nmol/g/min)

ÃŸ-hydroxybutyrate

10 15 20 25 30 35availability Inmol/g/min)

release(nmol/g/min)

10-

20

10 20 30 40 50 60 70availability (nmol/g/min)

release (nmol/g/min]

Fig. 6. 0-Hydroxybutyrate (top) and acetoacetate (bottom) uptake or releaserates per unit weight of medullary (â€¢)and squamous cell (O) breast cancerxenografts (/3-hydroxybutyrate: medullary, y = 0.525* - 1.204, 2/> < 0.001;squamous cell, y = 0.549* - 0.417, IP < 0.001; acetoacetate: medullary, y =0.296* - 2.876, 2P< 0.001; squamous cell, y = 0.219* - 0.758, 2P< 0.01).

nutritive tumor blood flow. The uptake of /3-hydroxybutyralecombined wilh lhal of aceloacelale was linearly relaled lo Iheoxygen consumption of the breasl cancer xenografts (IP <0.001). The ralio of ÃŸ-hydroxybulyrale and aceloacelale in Ihelumor-venous blood is assumed lo be an indicalor of Ihe inlra-milochondrial redox slale (10). This ralio was low in smalllumors (tumor size below 1 g) wilh high flow values and shiftedtowards higher values as tumor blood flow was reduced aladvanced growlh stages. This indicated an enlargemenl of hy-poxic areas as Ihe breasl cancer xenografts increased in size.Similar resulls have been oblained wilh O2-sensilive microelec-irodes using s.c. transplants of the same human breast cancerlines (21).

Spatial Distribution of Glucose, LÃ¡clate,and ATP Concentrations in s.c. Breast Cancer Xenografts. Glucose, lÃ¡clale, andATP levels were heterogeneously distribuled in medullary andsquamous cell lumors as compared wilh normal lissue (Fig. 7),and exlended areas with very low substrate concentralions(almosl al Ihe background level) were obvious in Ihe lumorlissue. The dislribulions of single substrates in successive sections of the same tumor were very similar. However, no correlations belween Ihe dislribulion profiles of glucose, lÃ¡clale orATP measured in conseculive lissue slices were delecled indicaling complex interrelationships belween Ihe substrate supply,Ihe removal of melabolic wasle producÃsand Ihe energy statusof malignanl lumors.

Tissue pH Measurements in s.c. Breasl Cancer Xenografts.7267




Fig. 7. Spatial distribution of glucose (A, D). lÃ¡clate(B, E) and ATP (C. F) concentrations registered in consecutive sections of a human medullary breast cancerxcnograft ( I ( : wet weight: 2.4 g) and of rat skeletal muscle (/â€¢>/).The values are obtained relative to the maximal concentration in the experimental series. Thehigh lÃ¡clateconcentrations in the skeletal muscle are attributed to twitching activities prior to the biopsy procedure. According to in vitro calibration studies (29), thehighest ATP concentrations correspond to approximately 2.0 HIM.

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pH distributions were investigated in 36 medullary and 24squamous cell breast cancer xenografts. Relevant systemic parameters of the host animals as well as statistical data on themeasured tissue pH values are listed in Table 2. Compared tos.c. tissue at the abdominal wall of nude rats, the pH distributions in both types of human mammary carcinomas were shiftedtowards more acidic values (Fig. 8, 2P < 0.001). In the squamous cell breast cancers higher pH values were found than inthe medullary tumors (2P< 0.001). The appearance of necroticareas was noted in the squamous cell tumors at earlier growth

Table 2 Mean arterial blood pressure (MABP), respiratory gas parameters withinthe arterial blood, and relevant parameters of the statistical evaluation of

measured tissue pH values

Values are means Â±SE.

s.c. human breast cancerxenografts

No. ofexperimentsNo.of pHreadingsBody

weight(g)Tumorgrowth period(days)Tumorwet weight(g)MABP

(mmHg)ArterialpO2(mmHg)ArterialpCO2(mmHg)Arterial

pHArterialSo2" (Sat.%)Arterial

hematocrit(v/v)MeantissuepHMedian

tissuepHModalclassRangeMedullary

carcinoma362633233

Â±534Â±14.7Â±0.5124Â±184

Â±239Â±17.38Â±0.0195Â±10.42Â±0.016.81

Â±0.046.826.8-6.95.78-7.59Squamous

cellcarcinoma241580198

Â±1031Â±12.8Â±0.3124

Â±382Â±237

Â±17.39Â±0.0196Â±10.46Â±0.027.04Â±0.047.037.0-7.16.51-7.62Control

(subcutis)2182221

Â±8125

Â±280Â±139

Â±27.39Â±0.0196Â±10.44Â±0.017.26Â±0.047.287.3-7.46.97-7.95

*Soi, O2 saturation of hemoglobin.

frequency (%)

Â¿.0-

20-

20-

10-

0::

20-

10-

subcutisNU21,n =8

medullary breast caN=36,n=2633

squamous cell breast ca.

5.5 6.0 6.5 7.0 7.5 8.0

tissue pH valueFig. 8. Cumulative frequency distribution (histograms) of pH values measured

in the subcutis of nude rats (A) and in medullary (B) and squamous cell breastcancers (C). Broken line, median pH value. N = number of animals or tumors,respective, n = number of measurements.

7269

stages than in the medullary cancers (21). In both tumor types,marked intertumor pH variations were observed. A trend wasfound towards an alkalization of the median tumor pH withincreasing tumor size which was statistically not significant.The pH values measured within single tumors exhibited veryheterogeneous distributions with large pH gradients in sometumors and very flat pH profiles in others.

DISCUSSION

The "tissue-isolated" implantation of human tumor xeno

grafts permitted for the first time the detailed and systematicinvestigation of substrate turnover by human tumor tissuesunder well-defined systemic conditions. So far, no strikingdifferences on substrate turnover of xenografted human breastcancers compared to isotransplanted rodent tumors are obvious.Data on blood flow and oxygen consumption of primary humanbreast cancers as compared with breast cancer xenografts indicate that functional characteristics regarding blood flow andoxygen supply are retained during xenotransplantation (21).The usefulness of "tissue-isolated" tumor models for the inves

tigation of tumor metabolism has been demonstrated previouslyusing rodent tumor transplants (2-12, 33, 34). Besides theinvestigations described, the model may be used for the detailedanalysis of other substrates, for the investigation of intratumorpharmacokinetics of anticancer agents and for the determination of production rates of different tumor-specific substances(e.g., tumor angiogenesis factor, a-fetoprotein or carcinoem-

bryonic antigen). For such measurements, the concentrationsof the substrates of interest have to be obtained with suchaccuracy as to permit the calculation of arterial-tumor-venousconcentration differences. The nude rat was chosen as tumorhost since its size permits experiments under well-defined physiological conditions. However, the usefulness of these animalsis limited by a possible host-versÂ«i-graft reaction which startsabout 4 months after birth. Since meaningful data can only beobtained before the onset of this reaction, relatively fast growinghuman tumors had to be chosen.

In the present study, the glucose consumption of xenograftedmedullary and squamous cell breast cancers was high, beingcomparable to brain tissue (0.25-0.70 /Â¿mol/g/min, 35, 36).This might be explained by changes of enzyme patterns occurring in transformed cells during carcinogenesis. Immortalization and transformation lead, among other changes, to anincreased glycolytic and glutaminolytic capacity. In this way,malignant cells are able to meet the increased energy requirements caused by the enhanced nucleic acid and phospholipidsynthesis (37). These metabolic changes are brought about bythe induction of isoenzymes different from those expressednormally (38-43). As a result, the glycolytic capacity both inthe presence and absence of oxygen is elevated under in vitroconditions.

Data on human tumor tissue in vivo are scanty. Measurements of arterial-tumor-venous concentration differences ofsarcoma-bearing limbs (14) and head and neck cancers in patients (15) indicated an increased glucose utilization by humantumors as compared with tissues at the site of tumor growth.Since tumor blood flow values were not determined in thesestudies no conclusions can be drawn regarding the actual glucose consumption of the tumors investigated. Using noninva-sive PET techniques (44) glucose uptake of various lung tumors was found to be 8 times higher than tumor-free lungtissue. The mean glucose consumption rates of xenograftedhuman breast cancers were well within the range of values




reported earlier for various rodent tumors (range, 60-888nmol/g/min; 4, 7, 9-12, 45). Similar to oxygen consumption(21), glucose uptake rose with increasing availability, indicatingnutritive tumor blood flow to be the rate-limiting factor in thesetumors. Similar results have been reported earlier for iso-transplanted rodent tumors (4, 7, 9-12). The direct relationshipof oxygen consumption and glucose uptake which was foundfor human breast cancer xenografts further suggests that bothrespiration and glycolysis were governed by nutritive blood flowas previously indicated in studies on rodent tumors (5). Sincetumor blood flow decreased significantly at advanced tumorgrowth stages, a reduced glucose uptake is expected at largertumor sizes. An increase of the glycolytic rate from 50 to 100%indicates an enlargement of hypoxic tumor areas with maintained glucose supply (46). This finding is supported by theoretical analyses of supply radii around tumor microvesselstaking into account actual in vivo data (46).

Theoretically, in tumors with adequate nutritive flow maximum glucose uptake should occur which corresponds to theglycolytic capacity of the tumor cell line investigated. Thus,"saturation" of the glucose uptake should be encountered at

very high availabilities and therefore, a hyperbolic relationshipof glucose consumption and supply has to be expected. Such arelationship is substantiated by fitting the data to the followingformula

glucose supplyA'm+ glucose supply (D)

where ^Â«m = actual glucose consumption, Fgi-maximai= maximal glucose uptake, and A',,, = glucose availability at half-

maximal glucose uptake.In medullary breast cancer xenografts, Fg|.maximaiis expected

to be 1.57 Â¿imol/g/min (Fig. 9, top; average A'm,3.46 /Â¿mol/g/

min) and 0.76 ^mol/g/min in squamous cell carcinomas (Fig.9, bottom; mean A'm, 1.20 ^mol/g/min). Such an analysis may

permit the evaluation of inherent differences of the glucosedemand of different tumor cell lines in situ.

LÃ¡clateis assumed to be mainly a glycolytic product since adirect relationship between glucose consumption and lactateproduction was found. However, a lactate release exceeding theamount that could be produced even at maximal glycolytic ratesfrom the amount of glucose utilized indicates other sources forglycolysis, e.g., amino acids. In vitro studies (35) and some invivo results (9, 10) suggested that glutamine might be the mostlikely precursor for the excess lactate production. However, thiswas not the case in the present study since there was a netglutamine release from these breast cancer cell lines (46). Thisfinding has to be expected considering that (a) oxygen is necessary for the oxidative breakdown of glutamine to pyruvate("glutaminolysis"; 47, 48) and (b) large areas with hypoxia and

even anoxia are present in subcutaneous breast cancer xenografts from the cell lines used here (21). Ketone bodies werepredominantly taken up by the xenografted tumors. Ketonebody uptake by breast cancers is not surprising since thispathway can also be activated in the mammary gland duringlactation for milk synthesis (49). However, under conditions oflow fumarate levels, acetoacetate formation has been reportedfor mammary gland homogenates (50). In some breast cancers,this mechanism could aggravate the tissue acidosis observed.

Ketone body utilization was demonstrated previously in headand neck cancers (15), and for a variety of rodent tumors (9,51). In the present study, the uptake rates of both ÃŸ-hydroxy-butyrate and acetoacetate were related to the respective supplyvalues, suggesting that the capacity of human breast carcinomas

01234

Glucose supply (|Uinol/g/min)Fig. 9. Glucose uptake rates of medullary (top, â€¢¿�)and squamous cell breast

cancer xenografts (bottom, D) as a function of glucose availability emphasizingsaturation of glycolytic activity at adequate supply rates (e.g., high nutritiveperfusion) of tumors in vivo.Broken lines, the respective maximum glucose uptakerates. Half-maximal glucose uptake rates and the corresponding glucose supplyrates are marked with arrows.

to consume ketone bodies is substantial. However, the overallcontribution of ketone bodies to the energy status of breastcancer xenografts is probably minimal due to the small amountabsorbed as compared with glucose. Nevertheless, ketone bodyturnover may be monitored as an indicator of the tumor oxygÃ©nationstatus. This is indicated by changes of the 0-hydroxy-butyrate/acetoacetate-ratio with increasing tumor wet weightleading to reduced nutritive blood flow and oxygen availabilityper unit weight at advanced growth stages.

The spatial distributions of glucose, lactate, and ATP concentrations were very heterogeneous due to the nonuniformdistribution of nutritive blood flow (13). The lactate productionwas governed by glucose uptake, which in turn depended onglucose availability and oxygen consumption. From these substrates and possible other sources, ATP is formed to meet theenergy requirements of rapidly growing cancer cells. Thus, theATP concentration depends on the substrate supply as well ason its rate of metabolic breakdown. Noninvasive measurementsusing nuclear magnetic resonance and positron emission tomography indicated that the energy status of malignant tumorsis mainly governed by tumor size (52, 53). However, for theassessment of tumor response to treatment, the evaluation ofchanges of the energy status in microregions of malignanttumors might be superior to integrated values over the wholetumor mass (54).

The pH values obtained in human mammary carcinoma7270




xenografts are comparable to those found earlier in a variety ofrodent and human tumors (31). The tissue acidosis is broughtabout by severe restrictions of nutritive blood flow at advancedgrowth stages, by high aerobic and anaerobic glycolysis and byreduced removal of metabolic waste products, predominantlylactic acid (13). The significantly higher pH values in squamouscell carcinomas coincided with the appearance of regressivechanges and necrosis at early growth stages (21). In tissue areaswith established necrosis, glycolysis ceases and conformationalchanges of proteins with exposure of proton-binding structuresoccur. This alleviates tissue acidosis (13, 31). On the otherhand, the squamous cell breast cancers exhibited a high ammonia production which could shift tissue pH to more alkalinevalues. This mechanism could also be responsible for the earlyonset of necrosis (55).

The weight-related decrease of tumor blood flow in humanmammary carcinomas with increasing tumor wet weight (21) isnot accompanied by a significant drop in the interstitial pH,although an impaired intramitochondrial energy production inthese tumors is evident with increasing tumor hypoxia. Obviously, the severe restrictions of convective and diffusive transport with progressive tumor growth led to a decrease of glucoseavailability and, subsequently, to a decrease of glucose turnover.As the lÃ¡clateproduction usually correlated well with the glucose consumption one may conclude that the drop of tumorblood flow led to a restriction of the substrate available forglycolysis. Therefore, increasing tumor wet weight must notnecessarily be accompanied by a continual pH drop.

From the results obtained it is concluded that in the breastcancer xenografts investigated nutritive tumor blood flow butnot the metabolic demand of the cancer cell determines theglucose uptake, the lÃ¡claterelease, and the kelone body lurn-over. The spadai dislribulion of glucose, lÃ¡clale, and ATPwithin tumor microregions is very inhomogeneous due to anonuniform distribulion of tumor perfusion within Ihe lumormass. Tissue acidosis is a resull of pronounced microcirculaloryand melabolic alterations during tumor growth.

ACKNOWLEDGMENTS

Professor Dr. H. Gabbert (Institute of Pathology, University ofMainz, Mainz, FRG) performed the histolÃ³gica! examination of thehuman breast cancer xenografts. The skilled assistance of K-H. Schlen-ger, Dipl. Biol., (Department of Applied Physiology, University ofMainz, FRG) in the statistical data analysis is highly appreciated. Dr.P. Okunieff, Department of Radiation Medicine, Massachusetts General Hospital, Boston, is gratefully acknowledged for his careful linguistic help and his support in performing nonlinear curve fitting.

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1988;48:7264-7272. Cancer Res F. Kallinowski, P. Vaupel, S. Runkel, et al. Cancer Xenografts in Nude RatsMetabolic Micromilieu, and pH Distributions in Human Breast Glucose Uptake, Lactate Release, Ketone Body Turnover,

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