[CANCER RESEARCH 46, 1427-1432, March 1986]

In Vivo 31P-Nuclear Magnetic Resonance Study of the Response of a Murine

Mammary Tumor to Different Doses of 7-Radiation

P. E. Sijens,1 W. M. M. J. Bovée,D. Seijkens, G. Los, and D. H. Rutgers

Departments of Radiotherapy [P. E. S., D. H. R.] and Pathology [G. L.], University Hospital Utrecht; Department of Applied Physics, Delft University of Technology[W. M. M. J. B.]: and Department of Organic Chemistry, University of Utrecht [D. S.], The Netherlands

ABSTRACT

The response of the s.c. implanted murine mammary carcinoma NU-82 to 10 and 20 Gy of ^-radiation was followed by invivo 31P-nuclear magnetic resonance spectroscopy on 48 mice

during a period of 2 days. During the first 8 h after a dose of 10Gy, the ratio of ATP/inorganic phosphate increases to a value of120 ±15% (SE) of the control value. This rise is followed by adecrease of the ratio to 74 ±12% after 47 h. However treatmentwith 20 Gy causes the ratio of ATP/P¡to decrease gradually toa level of 45 ±7% after 47 h. Histológica! investigations demonstrated that radiation-induced necrosis largely contributes to

this phenomenon. During the time of observation irradiated tumors displayed no changes in size and intracellular pH. Aftertreatment with 10 Gy as well as with 20 Gy, the phosphodiesterresonance decreased in intensity. By 31P-nuclear magnetic res

onance spectroscopy on extracts of the NU-82 tumor, twophosphodiesters were identified, glycerophosphocholine and gly-

cerophosphoethanolamine.

INTRODUCTION

Phosphorus NMR2 spectra can provide information regarding

the cellular phosphate compounds which participate in tissuemetabolism. Localized high-resolution 31P-NMR spectra can beobtained in animals and humans. Thus 31P-NMR spectroscopy

may be used to study metabolic disorders caused by pathologicalprocesses like cancer. As compared with other tissues, tumor31P-NMR spectra show high levels of PME and P¡.During growth

tumors become more hypoxic and necrotic, which results inincreased levels of P¡and decreased levels of the high-energyphosphates (1-9). During this process tumors may show someacidification, up to 0.5 pH unit (6-10). The greatest potential of31P-NMR spectroscopy in oncology is supposed to be in permit

ting noninvasive monitoring of the response of tumors to varioustherapeutic modalities. Chemotherapy and hyperthermia havebeen shown to produce distinct and substantial changes in 31P-

NMR spectra (5, 7-9, 11-15). Evanochko ef al. (5, 7) and Ng ef

al. (9) reported the effects of 14 Gy radiotherapy on a murinemammary tumor. The tumors of the 5 mice they treated showedchanges in PCr, the high-energy compound which before treat

ment was about equal in intensity to the ATP resonances; after

Received 6/13/85; revised 10/24/85; accepted 10/25/85.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.

1To whom requests for reprints should be addressed, at the Department of

Radiotherapy, University Hospital, Catharijnesingel 101, Utrecht, The Netherlands.2The abbreviations used are: NMR, nuclear magnetic resonance; PME, phos-

phomonoester; PDE, phosphodiester; PCr, phosphocreatine; NTP, nucleoside tri-phosphate; DPDE, diphosphodiester.

a reproducible disappearance within the first 15 min after therapy, they found a recovery of PCr, in one case of five, clearlybeyond the pre-treatment level and the intensities of the ATP

resonances. This effect, which occurred within 1.7 days, wasaccompanied by a 50% decrease in tumor mass. The sameauthors also investigated the effect of 14 Gy radiotherapy on aradiation induced fibrosarcoma (5). They found some tumors toexhibit an increase of PCr which was accompanied by a neartotal disappearance of the P¡peak, but others showed a largereduction in PCr during the first days after irradiation. Skog efa/. (16) investigated the effect of 5 Gy X-radiation on Ehrlichascites tumor cells in mice. Their in vitro 31P-NMR spectra

showed no changes during the first 48 h after treatment, exceptfor a temporal 50% reduction in ATP/P¡ratio at 20 to 24 h. Weused 31P-NMR spectroscopy to study the response of a solid

murine mammary tumor to 10 and 20 Gy doses of 7-radiation.

In this paper we report our findings, which contrast with thoseof Evanochko ef al. (7) and Ng ef al. (9).

MATERIALS AND METHODS

Tumors. Measurements were performed on tumors from female mice.The mice were from the DBA-2 strain and were approximately 5 monthsold. Pieces of the NU-82 mammary tumor, which spontaneously arosein a female DBA-2 mouse, were transplanted s.c. on the flank. For in

vivo NMR measurements mice were anesthetized with low doses ofValium/Hypnorm (8 mg fluanison, 0.16 mg fentanyl base, and 5 mgdiazepam/kg i.p.).

Radiotherapy. Total-body irradiation of mice was performed withphotons from a ^Co source with a dose of 10 or 20 Gy (dose rate, 0.85

Gy/min). The mice were treated in a cage with a height of 2 cm, coveredwith a lid of 2 cm Perspex to establish equilibrium conditions within thecage. For control of some mice only the tumors were irradiated byshielding the remainder of the (anesthetized) animals with blocks of lead.The 31P-NMR measured metabolic response of their tumors was found

to be identical to the tumor response of the totally irradiated mice.Nuclear Magnetic Resonance in Vivo. All in vivo 31P-NMR spectra

were measured on a home-built 7 T spectrometer (17) operating in theFourier transform mode. We used the following spectral parameters: 45°

flip angle at the center of the coil, 2K data points with quadraturedetection, and 1.0 s recycle time. Resolution was enhanced by theconvolution difference method (filter width, 1.3 KHz and line broadening,25 Hz). Our tumor spectra are the sum of 400 or 600 scans (total scantime, 6.7 or 10.0 min). Tumors were measured using 2-turn solenoidal

coils, the remainder of the mouse being shielded by a Faraday shield asdescribed by Evanochko ef al. (5). The assignments of resonances toPME, P¡,and PDE were made by 31P-NMR analysis of tumor extracts

(see next paragraph). The assignments of the remaining resonances toPCr, ATP, and DPDE were made from literature values. The apparent¡ntracellularpH was estimated from the chemical shift of the P¡resonanceusing the calibration data of Moon and Richards (18). Relative concentrations were estimated from the integrated areas of the corresponding31P-NMR resonances.

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By measuring a mouse without a tumor which was positioned in theprobe in the same manner as the tumor-bearing ones, it was ascertainedthat underlying tissues don't contribute to our tumor NMR spectra.

Nuclear Magnetic Resonance in Vitro. In vitro31P-NMR spectroscopy

was performed upon extracted material to identify unknown resonances.After the mouse had been anesthetized with diethyl ether, the tumorwas quickly removed and put into a stainless steel tube which was filledwith liquid nitrogen. While submerged in the nitrogen, the tumor wasground into a fine powder using a steel stave which was also precooled.The frozen power was treated with 2 volumes of 7% perchloric acid,centrifuged, neutralized, and centrifugea again to remove the KCIO4precipitate. After freeze-drying the residue was dissolved in a 100 mwi

EDTA solution which contained 20% D2O. In vitro NMR spectra weremeasured on a 4.7 T spectrometer operating at 81.0 MHz in the Fouriertransform mode. We used the following spectral parameters: 45° flip

angle, 8K data points with quadrature detection, and 1.0s recycle time.Resolution was enhanced by the convolution difference method (filterwidth, 6.3 kHz and line broadening, 2.5 Hz). The spectrum, which isshown in Fig. 1£>,is the sum of 1024 scans (total scan time, 17 min).

Histology. S.c. tumors were removed, fixed in 4% formaldehyde, andembedded in paraffin. Tissue sections were stained with hematoxylin-

eosin. By means of a camera the images of the tissue sections wereprojected from a light microscope upon a graphic tablet. Subsequentlythe surface areas of necrotic and vital tumor tissue were determinedmorphometrically using a manual point counting technique with thegraphic tablet linked to a microprocessor minicomputer.

io.o o.o -io.o -20.0

PPMFig. 1. In vivo 31P-NMRspectrum of a NU-82 tumor (a) and the spectrum of a

perchloric acid extract which was made from 2 tumors NU-82 (i>). The in vivospectrum, which is the sum of 400 scans, shows the resonancesof PME (peak 1at 3.8 ppm relative to 85% phosphoric acid), P,(2:2.0 ppm), PDE (3:0.2 ppm), PCr(4:-3.1 ppm), i-ATP (5:-5.5 ppm), «-ATP(6:-10.7 ppm), DPDE (7:-12.8 ppm)and fi-ATP (8:—19.2ppm). The extract spectrum, which is the sum of 1024 scans,shows PME (peak 1) and PDE(peak 3) split into, respectively,3 and 2 resonances.

RESULTS

The 31P-NMR spectrum of the mammary carcinoma NU-82 as

passaged in a syngeneic population of mice is characterized byhigh levels of PME, P¡,and NTP and low levels of PDE, PCr, andDPDE (19). Because ATP has been reported to be the majortumor NTP compound (20, 21) and because, thermodynamicallyspeaking, UTP, GTP, and CTP are considered to be identical toATP (22), in this paper we refer to the nucleoside triphosphatesas ATP. The in vivo spectrum shows 8 resonances (Fig. 1a). Inagreement with the finding that all in vivo NMR signal arises fromthe tumor (see "Materials and Methods"), the extract spectrum

shows the same resonances, including PCr (Fig. 1b). In theextract spectrum, however, the resonances of PME (peak 1)and PDE (peak 3) are split into, respectively, 3 and 2 peaks.

By addition of organic phosphates to tumor extracts andcareful analysis of the pH titration curves (using data from Refs.23-26) the major PME resonances (at 3.7 and 3.1 ppm relative

to 85% phosphoric acid) and both PDE resonances (at 0.4 and-0.1 ppm) were identified as, respectively, phosphoethanola-mine, phosphocholine, glycerophosphoethanolamine, and gly-

cerophosphocholine. This is in agreement with the tumor peakassignments of other authors (5, 20, 27).

In vivo31P-NMR spectra of mammary carcinomas NU-82 show

a considerable degree of variation due to individual differencesin growth rate, resulting in tumors of different size and stage ofgrowth. The average tumor volume doubling time amounted to5 days. Fig. 2 illustrates the influence of tumor size on the 31P-

ppmi 0-10-20

Fig.2. In vivo 31P-NMRspectra of different NU-82tumors 20 days after implantation. Tumor a (mass, 0.7 g) was measured using a solenoidal coil of 12 mmdiameter; tumors b (mass,2.0 g) and c (mass, 3.4 g) were measuredwith a coil of17 mm. In each stage of growth, the pH, as determined from the chemicalshift ofthe P¡resonance, is equal to 7.2. Eachspectrum is the sum of 400 scans. Spectralassignments are as in Fig. 1.

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NMR spectrum of mammary carcinoma NU-82. To a lesser extent

tumors of the same size also showed some degree of variation.The spectrum of each individual tumor was highly reproducible,and during 48 h of untreated growth no spectral changes occurred.

We investigated the metabolic response of the solid murinemammary tumor NU-82 to 10 Gy of 7-radiation by measuring it

beforehand and at 4, 8, 26, and 47 h after irradiation. Thustumors of 23 mice with an average tumor mass of 1.5 g (compareFig. 2o) were measured at 20 to 22 days after implantation.Because of the limited availability of NMR measuring time, mostanimals were measured at three of the five points of time whichare mentioned above. For this tumor, which is characterized bylow levels of PCr, we judge the ratio between the high-energyphosphate ATP and the low-energy phosphate P¡as a suitable

indicator of metabolism. As a matter of fact, we found significantchanges in the ratio of ATP/P¡as determined from the integralsof the resonances of 0-ATP (peale 8), the resonance of ATPwhich doesn't overlap with ADP or DPDE, and P¡(peak 2). To

eliminate variations in ATP/P¡ratio among different untreatedtumors, the following values are normalized to the level beforetreatment being 100%, with standard errors of the mean (4 «n« 14) determined from individual changes after treatment. Adecrease to 73 ±6% (SE) at 4 h after treatment with 10 Gy isfollowed by an increase to 120 ±15% at 8 h and a decrease to53 ±7% at 26 h and 74 ±12% at 47 h (Fig. 3, open circles). Atthe same points of time the tumors of 25 mice were measuredafter radiotherapy with a dose of 20 Gy. Contrary to the resultsat 10 Gy, after 20 Gy of 7-radiation the ratio of ATP/P, shows acontinuous decrease to 45 ±7% of the pre-treatment value at

47 h after treatment (Fig. 3, closed circles). In the discussion wewill deal further with the dose-response relationship regarding

the changes in ATP/Pi ratio after 10 and 20 Gy. Here, still, someremarks will be made about observations which apply to the

150- -

-O— 10 Gy

—•20 Cy

100

50- -

therapeutic response of the tumors within the first 47 h aftertreatment with 10 Gy, as well as after treatment with 20 Gy of7-radiation (a) no change in tumor volume occurs, while un

treated tumors show some 30% increase during 2 days ofgrowth; (b) intracellular pH, as determined from the chemicalshift of the P¡resonance, remains equal to 7.2. The chemicalshifts of the other 31Presonances are also unchanged; and (c)

the concentration of PDE (mainly glycerophosphocholine, peak3) decreases. The spectra of a tumor before (Fig. 4a) and at 8 hafter treatment (Fig. 4o) clearly show this (see Fig. 4). However,because the PDE resonance usually is less well resolved, after48 series of in vivo measurements we still judge it too early toquantify this radiation effect.

To investigate whether the response to irradiation we observed is specific for tumors, we also investigated the hind legat different intervals after radiotherapy. After total-body irradiation of healthy as well as tumor-bearing mice, the 31P-NMR

spectrum of hind leg muscle was unchanged between 4 and 47h after treatment.

DISCUSSION

The metabolic response, as measured by 31P-NMR spectros-

copy, of mammary carcinoma NU-82 to 10 Gy of cobalt 7-

radiation differs from the response to 20 Gy. After 20 Gy theATP/Pi ratio almost linearly decreases during the first 2 daysafter treatment (Fig. 3, closed circles). The 10 Gy responsefollows a more complicated pattern (Fig. 3, open circles). TheStudent's i-test was applied to evaluate the statistical signifi

cance of the changes in ATP/P, ratio after a dose of 10 Gy ascompared with the changes after 20 Gy. While at 4 h aftertreatment the 10 Gy ATP/Pi ratio is lower than the 20 Gy ATP/P¡ratio [73 ±6% versus 92 ±6% (P < 0.05)], at 8 h the 10 Gyratio is higher than the 20 Gy ratio [120 ±15% versus 85 ±10% (P < 0.1)]. At 26 h after treatment there is no difference(53 ±7% versus 60 ±8%), but after 47 h the 10 Gy ratiois significantly higher again [74 ±12% versus 45 ±7% (P <

Time after irradiation (hours)

Fig. 3. Ratio of /J-ATP/P¡after radiotherapy with a dose of 10 (O)and 20 Gy (•)as a percentage of the value before treatment, plotted against the time. Bars, SE.The number of determinations per graphic point varied between 4 and 13.

0 -10 -20Fig.4. In vivo 31P-NMRspectra of one NU-82tumor before (a) and at 8 h after

20 Gy irradiation(b). Both spectra are the sum of 600 scans. Spectral assignmentsare as in Fig. 1.

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Wl*-•^P^'^Är^;^v.,^&'^,,..^^

Fig. 5. In vivo 31P-NMR spectra of a necrotic tumor NU-82 (6) (n = 0.90, ATP/P, = 0.16) and a less necrotic one (A) (n = 0.49, ATP/P, = 0.54) with the correspondinghematoxylin-eosin stained histological sections (N, necrotic tumor tissue; V, vital tumor tissue). Both NMR spectra are the sum of 600 scans. Spectral assignments areas in Fig. 1. Magnification, x 125.

0.025)]. However the responses of tumor NU-82 to 10 and 20

Gy irradiation have in common the clearly reduced ATP/Pi ratioof 26 and 47 h after treatment. A possible involvement of somefactors in the changes of the ATP/P¡ratio after radiotherapy willbe discussed below.

(a) Increased ATP Consumption to Repair Radiation Damage. This effect may persist throughout our period of observation; Skog er al. (28) observed changes in the morphology ofEhrlich ascites tumor cells after irradiation with a dose of 5 Gy,up to 72 h after treatment. Regarding our own observations, thedecrease in the free concentration of the PDE (glycerophospho-

choline + glycerophosphoethanolamine) after both 10 and 20 Gyradiotherapy may be caused by increased incorporation of thatphosphate compound to restore damaged membranes.

(b) Decreased ATP Production by Conversion from AerobicMetabolism to Glycolysis. It is well known that aerobic tumorcells are more radiosensitive than are hypoxic cells. Since afterradiotherapy murine tumors are known to reoxygenate withinabout 6 h (29-31), this radiation-induced metabolic effect may

be limited to the first hours after treatment. As tumor glycolysishas been reported to be coupled to acidification of the tissue(32-38), our observation of the tumor pH remaining equal to 7.2

after radiotherapy does not support an involvement of this factorin our observations.

(c) Fluctuations in ATP Level by Cell Cycle Synchronization. Ishiguro ef al. (39) and Skog ef al. (40,41 ) reported elevatedATP levels in tumor cells being in the post-synthetic gap (G2),

the cell stage in which irradiated tumor cells accumulate. Usingflow cytometry we observed no significant changes ¡nthe cellcycle distribution except some elevation of G2 at 8 h after a doseof 10 Gy, from 2 ±0.5% to 5.1 ±0.5%. However, unless in theG2 phase of tumor NU-82 the ATP level with respect to the P,

level is one order of magnitude higher than in the other phases,the observed G2 accumulation cannot explain the NMR measured elevation of the ATP/P¡ratio at 8 h after a dose of 10 Gy.

(d) Death of Tumor Cells. After death organic phosphates,which are normally seen in the high-resolution 31P-NMR spec

trum, are broken down to P¡.Four untreated tumors NU-82 (ofdifferent size) which had been measured by in vivo 31P-NMR

spectroscopy were investigated histologically to correlate tumornecrosis with the ratio of ATP/P¡.By staining different tissuesections with hematoxylin-eosin, 4 determinations of necrosis

were performed on each tumor. The thus calculated mean fractions of necrosis diverged from 0.49 to 0.90, depending on the

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tumor of investigation. These values have standard errors of themean of about 4%. The necrotic fraction of the tumor (n) wasfound to correlate linearly with the NMR determined ATP/P¡ratioaccording to the equation: n = 1 - 0.72 (ATP/P¡).The equationmeets a linear coefficient of correlation of r = -0.90. In Fig. 5the 31P-NMR spectra of the least necrotic tumor (Fig. 5a) (n =

0.49, ATP/Pi = 0.54) and the one of the most necrotic tumor(Fig. 5b) (n = 0.90, ATP/Pi = 0.16) are shown together with

corresponding histological preparations. Two tumors were analyzed at 26 h after irradiation with a dose of 20 Gy (when theratio ATP/Pi amounted about 53% of the starting level; see thefirst section of "Discussion"). The histologically determined n-

values were in close agreement with the values which werecalculated using the equation: 0.77 versus 0.78 and 0.72 versus0.67. This finding indicates that radiation-induced changes in the

ATP/Pi ratio roughly reflect changes in the amount of necrosiswithin the tumor.

We conclude that radiation-induced necrosis (point d) largely

contributes to the observed spectral changes. In order to assessthe relative importance of membrane repair (point a) and glycol-

ysis (point b), and in particular also to explain the complicatedpath after a dose of 10 Gy and its relation to the cell cycle (pointc), ¡nthe future we will continue to support our 31P-NMR studieswith pathological research and start 'H-NlvIR investigations.

Our 10 Gy ATP/Pj pattern resembles the results of Skog ef al.(16), who irradiated Ehrlich ascites tumor cells with a dose of 5Gy and found a temporal 50% reduction in the ATP/P¡ratio at20 to 24 h after treatment. Our finding that within the first 2 daysafter 10 and, in particular, 20 Gy of radiotherapy the high-energy

phosphate ATP (actually NTP) decreases (PCr is low from thebeginning) contrasts with the increase in PCr which Evanochkoet al. (5, 7) and Ng ef al. (9) found after 14 Gy radiotherapy totheir murine mammary tumor. However their radiosensitive tumor, which lost one-half of its weight within 2 days after treatment, differs from our mammary tumor NU-82, which, more in

agreement with the clinical practice, kept a constant weightduring the first 2 days after radiotherapy. In summary, this studyindicates that 31P-NMR spectroscopy may be used to measure

radiation-induced necrosis before changes in tumor volume oc

cur.

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1986;46:1427-1432. Cancer Res   P. E. Sijens, W. M. M. J. Bovée, D. Seijkens, et al.   -Radiation

γResponse of a Murine Mammary Tumor to Different Doses of P-Nuclear Magnetic Resonance Study of the31 In Vivo

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