Fractionated Radiotherapy of Rat ProstaticAdenocarcinoma (Dunning R3327-PAP) in

Nonanesthetized Animals

Torvald Granfors,1* Anders Bergh,2 Per-Olov Lofroth,3 and Anders Widmark4

1Department of Urology and Andrology, Umeå University, Umeå, Sweden2Department of Pathology, Umeå University, Umeå, Sweden

3Department of Radiophysics, Umeå University, Umeå, Sweden4Department of Oncology, Umeå University, Umeå, Sweden

BACKGROUND. The dose-response effect of fractionated external beam radiotherapy onnonanesthetized rats bearing the androgen-sensitive prostatic adenocarcinoma DunningR3327-PAP was studied.METHODS. The radiation was given with a photon beam from a 4-MeV linear accelerator indoses from 4 to 11 Gray per fraction during 5 consecutive days. When the tumors with lowand intermediate radiation doses relapsed into regrowth, the rats were castrated. Tumorvolumes and rat weights were followed, and at the end of the study a morphometric analysisof the tumors was done.RESULTS. Fractionated irradiation induced a dose-dependent delay in tumor growth inhormonally intact rats. Castration stopped the tumor regrowth, showing that some of thetumor cells were still hormone-sensitive. The study was facilitated by the nonanesthesiaprocedure.CONCLUSIONS. The Dunning R3327-PAP hormone-sensitive rat tumor is sensitive to ra-diotherapy in a dose-dependent way. Regrowing, irradiated tumors contain hormone-sensitive cells. This work provided basic knowledge for further experimental studies of theeffects of radiation on prostatic adenocarcinoma. Prostate 39:16–22, 1999.© 1999 Wiley-Liss, Inc.

KEY WORDS: Dunning R3327 rat prostatic adenocarcinoma; prostate cancer; radio-therapy; nonanesthetized rats

INTRODUCTION

External beam megavoltage radiotherapy is cur-rently used as a potentially curative form of treatmentfor clinically localized prostate cancer. Concerning thelong-term outcome of both radiotherapy and radicalprostatectomy, the most important parameters are thepretreatment T stage, histopathologic grade, andlymph node involvement of the tumor [1,2]. In recentyears, however, the curative potential of radiationtherapy in prostate cancer has been discussed due tothe high frequency of positive prostate biopsies afterradiotherapy, and results have been presented sug-gesting that a positive biopsy more than half a yearafter radiotherapy might indicate a higher risk of pro-

gressive disease [3–5]. Others have not found any sig-nificant correlation between positive biopsies andpoor prognosis [6]. Higher radiation doses would be amethod to sterilize the prostate gland from malignantcells, but previous studies have shown increasingcomplications with increasing doses [7], especiallywith doses above 70 Gy [8] using the conventional,opposed, four-field box technique [9]. Modern confor-

Grant sponsor: Swedish Society Against Cancer; Grant sponsor: Li-on’s Cancer Research Foundation; Grant sponsor: Medical Facultyof Umeå University.*Correspondence to: Dr. Torvald Granfors, Department of Urologyand Andrology, Umeå University, S-901 85 Umeå, Sweden.Received 10 September 1997; Accepted 29 September 1998

The Prostate 39:16–22 (1999)

© 1999 Wiley-Liss, Inc.

mal radiotherapy technique allows higher doses of ex-ternal beam radiation and is superior to conventionaltherapy [10]. Even doses above 80 Gy have been used,and dose-escalation studies of conformal radiotherapyare underway [11].

The androgen-sensitive Dunning R3327 prostaticadenocarcinoma mimics several features of the humanprostatic tumor. It is a relatively slow-growing, well-differentiated tumor that starts to regrow in an andro-gen-independent manner some time after castration[12–14]. Previous ionizing radiation studies on differ-ent variants of the Dunning R3327 tumor have showntheir sensitivities to single-dose irradiation [15–18],and the well-differentiated, hormone-sensitive Dun-ning-H tumor is more radiosensitive than the anaplas-tic, hormone-refractory Dunning-AT tumor [17].

The present dose-response study of fractionated ir-radiation on nonanesthetized rats bearing the hor-mone-sensitive prostatic adenocarcinoma tumor Dun-ning R3327-PAP was designed to investigate if the tu-mor could be eradicated or for how long a time tumorgrowth could be delayed.

MATERIALS AND METHODS

Animals

Tumor tissue from Dunning R3327-PAP rat prostat-ic adenocarcinoma was implanted as a 1-mm3 coresubcutaneously on both flanks of Copenhagen XFisher F1 hybrid male rats 10 weeks after birth. Eachrat was implanted with two tumors. This well-differentiated, androgen-sensitive tumor subline wasoriginally obtained from Dr. N.H. Altman (Organ Sys-tem Program of the National Cancer Institute, Miami,FL). The rats were bought from ALAB (Laboratoriet-janst AB, Sollentuna, Sweden) and housed in our ani-mal house in a controlled environment (12 hr light/12hr dark) and supplied with pelleted food and water adlibitum. The study was approved by the local ethicalcommittee for animal research.

Treatment

Only animals with bilateral tumors were chosen forthis experiment. The rats were stratified for tumor vol-ume, randomly allocated into four groups, and treatedas follows:

1. 4 rats, i.e., 8 tumors, control group = No treatment.2. 10 rats, 4 Gy × 5 to the left side tumor = 20 Gy.

5 Gy × 5 to the right side tumor = 25 Gy.3. 10 rats, 6 Gy × 5 to the left side tumor = 30 Gy.

7 Gy × 5 to the right side tumor = 35 Gy.

4. 10 rats, 9 Gy × 5 to the left side tumor = 45 Gy.11 Gy × 5 to the right side tumor = 55 Gy.

Radiotherapy

External beam radiation was performed 4 monthsafter inoculation of the tumors. The radiation wasgiven to each tumor with a vertical photon beam froma 4-MeV linear accelerator on 5 consecutive days. Inorder to avoid problems due to repeated anesthesia(use of anesthetics, hypoxia, animal death), we foundit possible to keep the nonanesthetized animal in ametallic frame by a strong cotton net during the ra-diation procedure. Two animals were oriented in op-posite directions and treated from below in the samefield. The dose rate was 2.5 Gy/min and the source-to-skin distance 70 cm. The radiation field size waschosen with at least a 5-mm margin around the pal-pable tumor. The animals were continuously observedthrough a video camera and in case they moved, theirradiation was immediately stopped and the positionof the tumor in the radiation field was checked andreadjusted, if necessary. In order to get an adequateradiation dose into the tumor, a 10-mm perspex platewas placed close to the animal and used to eliminatethe dose buildup, and on the exit side a 10-mm-thicksilicone slab was used above the animal to give back-scatter. Dosimetry controls were done before the treat-ment with diodes in a rat phantom of wax material inthe same geometry as for the irradiation of the rats.The rats used as controls were not treated with irra-diation, but were trained in the same frame for equallylong periods and were thus sham-treated.

Tumor Volumes

Tumor diameters were measured by the same in-vestigator (T.G.) with calipers on nonanesthetized ani-mals. Three perpendicular diameters were measuredprior to treatment and afterwards at 2–3-week inter-vals, and tumor volumes were estimated by the for-mula for an ellipsoidal mass, p/6 × D1 × D2 × D3. Thetumor volume on the first day of irradiation was de-fined as 100%. From that value the relative growth ofeach tumor was followed up regularly. When tumorsize had doubled from the start of treatment, that wasdefined as a relapse.

Castration

When the tumors with radiation doses between 20–35 Gy had clearly relapsed, these rats were castrated.The bilateral orchiectomy was performed through amidline scrotal incision under short ether anesthesia,

Fractionated Radiation of Dunning R3327-PAP 17

from which procedure the animals recovered within afew hours.

Morphological and Morphometric Analysis

At the end of the study the rats were sacrificed bydecapitation and three randomly chosen samples fromeach tumor were fixed in Bouin’s solution and embed-ded in methacrylate resin (Histo-resin, LKB, Stock-holm, Sweden). The tissue blocks were cut into 2-mm-thick sections and stained with hematoxylin and eo-sin. A morphological and morphometric analysis wasdone using light microscopy at ×400 magnification,and the volume densities of glandular epithelium, tu-mor stroma, and glandular lumina were calculated asearlier described [19].

RESULTS

Animals

The radiation procedure itself was well-tolerated bythe animals without anesthesia. They usually lay inpeace and quiet in the cotton nets for the few minutesrequired, and in case they moved that was immedi-ately noticed and the positioning of the tumor in the

radiation field could be readjusted, if necessary. Inpractice the tumors were seldom readjusted, becausethe middle part of the animal body was generallywell-fixed, although the animal could move its head,tail, and limbs to some extent. With the tumors placedin the limbs it would have been more difficult to per-form the experiment without anesthesia.

In the fourth treatment group (45 Gy to one sideand 55 Gy to the other side), the radiation dose wastoo huge for the rats to tolerate, and 7 of them werelost within the first month after the irradiation. Thusonly 3 animals in this group remained for the fol-low-up.

The changes of animal weights are presented in Fig-ure 1. Initially there was no significant difference inbody weight between the treatment groups (P = 0.21,Kruskall-Wallis one-way ANOVA). The treatmentprocedure induced weight loss, which in animals re-ceiving low and medium irradiation doses (20–35 Gy),started to return after 10 days. Weight loss was stillsignificant at 20 days as compared with weight at thestart. For animals receiving high irradiation doses (45–55 Gy), the weight loss was more prominent and didnot start to return to normal until after 20 days. Forsham-treated control rats there was no weight loss, but

Fig. 1. Mean animal weights in different treatment groups. Significant weight change as compared with weight at the start of treatmentis indicated by X = (P < 0.05) and XX = (P < 0.01) (Wilcoxon matched-pairs test). In the high radiation dose group there were only 3 animals,i.e., they were without statistical power in this test.

18 Granfors et al.

a slight weight gain from 20 days on after the start oftreatment, probably due to the natural growth of theseyoung animals.

Tumor Growth

The respective tumor volumes of the treatmentgroups at start and on days 90 and 130 are shown inFigure 2. At start there was no difference in tumorvolume between the treatment groups (P = 0.95,Kruskal-Wallis one-way ANOVA).

Irradiation delayed tumor growth as comparedwith the untreated tumors (Fig. 3). When tumorsstarted to regrow, a proportional dose-response wasobserved. At first tumors with low (20–25 Gy) irradia-tion grew faster than tumors with intermediate (30–35Gy) and high (45–55 Gy) irradiation doses. When themajority of tumors with low and intermediate irradia-tion doses had started to relapse at about day 130(Table I, Figs. 2, 3), the tumors in these treatmentgroups grew with equal velocity, i.e., the dose-response was not as obvious as during the initial ob-servation period. For the higher irradiation doses (45and 55 Gy), tumor regrowth was still significantly de-layed. Calculated with the Kruskal-Wallis one-way

ANOVA, there was a dose-dependent difference intumor growth from days 90–130 (P < 0.01), and thenwith lower probability to day 208 of follow-up (P <0.05).

The 20-, 25-, 30-, and 35-Gy treatment groups werecastrated on day 209 from start. These tumors wereapparently still hormone-sensitive, since the growth ofthe relapsing tumors slowed down after bilateral or-chiectomy for about 60 days (Fig. 4). Thereafter, a hor-mone-refractory tumor growth relapse was seen. Aproblem towards the end of this long-term study wasthe loss of tumors, because large cystic tumors rup-tured with skin penetration. These animals were sac-rificed and their tumors excluded from further calcu-lations. Only the growth of tumors followed to the endof the study are shown in Figure 4.

Morphology

The results of morphological studies on the tumorspecimens are shown in Table II. Only tumors fromthe endpoint of the study were analyzed. In the con-trol group the tumors were taken 90 days and in theother groups 286 days after the start of radiation treat-ment. Significance testing using Kruskall-Wallis one-

Fig. 2. Boxplot showing the tumor volume in mm3 on days 0, 90, and 130 from the start of various treatment regimens. The ‘‘box’’represents the interquartile range (from 25–75% of the values) with the median value marked inside, and the maximum and minimum valuesmarked outside. s, outliers, i.e., cases with values between 1.5–3 box-lengths from the upper edge of the box.

Fractionated Radiation of Dunning R3327-PAP 19

way ANOVA showed no difference between the stud-ied groups concerning the luminal compartment (P =0.17), but there was strong significance considering thedecrease of epithelial cells (P = 0.0002) and the in-crease of stromal tissue (P = 0.0001) with increasingradiation doses. Excluding the ‘‘no treatment’’ groupand comparing only the true treatment groups, thecorresponding figures were: P = 0.12 for the luminalcompartment and P = 0.004 for both the epithelial cellsand the stromal tissue. The higher the radiation dose,the fewer prostatic adenocarcinoma epithelial cellswere observed in the morphometric analysis. How-ever, in every tumor some epithelial cells were seen,and no total tumor eradication was found after ioniz-ing radiation in this series.

DISCUSSION

In contrast to previous studies which were per-formed on anesthetized animals [16–18], no anesthet-ics were used in this study, and the animals were keptin a special holder during the irradiation. The animalshad been ‘‘trained’’ in the holder a couple of timesbefore the start of irradiation and they did not seem tobe stressed, but lay quite peacefully during the shortprocedure. With this technique the risk of animaldeath is probably smaller, especially as the treatmentis repeated on 5 consecutive days. This way of han-dling the animals increases opportunities to performvarious fractionated irradiation studies.

Earlier studies with single-dose irradiation treat-

Fig. 3. Relative change of tumor volume (median) the first 208 days in intact control rats and in rats treated with five fractions ofirradiation up to 20, 25, 30, and 35 Gy (10 tumors in each group), and 45 and 55 Gy (3 tumors in each group), respectively.

TABLE I. Percentage of Tumors in Relapse During Follow-Up (Days), i.e.,Tumors That Had Doubled Their Original Size

18days

40days

62days

90days

130days

160days

208days

20–25 Gy (n = 20) 15 25 65 75 95 95 10030–35 Gy (n = 20) 15 20 35 50 60 90 9545–55 Gy (n = 6) 0 0 0 0 0 17 50

20 Granfors et al.

ment of the Dunning-H tumor showed that this tumoris radiosensitive. A single-dose treatment with 20 Gydelays regrowth up to about 100 days, and after thatthe tumors grow rapidly [17]. In the present study a5-day fractionated schedule was used, and the delay

of growth was similar to that shown earlier withsingle-dose treatments [17,18]. Initially a clear dose-response was observed after fractionated irradiationin a similar way as has been previously reported. Ahigh irradiation dose of 55 Gy had a favorable re-

Fig. 4. Relative change of tumor volume (median) during 286 days in intact control rats and in rats treated with five fractions of irradiationup to 20, 25, 30, 35, 45, and 55 Gy. Castration was performed on day 209 only to rats treated with 20, 25, 30, and 35 Gy.

TABLE II. Volume Densities of Tumor Stroma, Glandular Epithelium, andLumina After Different Treatment Modalities of Fractionated

Radiotherapy†

TreatmentNumber

of tumors Epithelium Stroma Lumina

No treatment 6 64.2 ± 3.1 27.0 ± 2.8 8.8 ± 0.820 Gy 4 48.0 ± 7.2* 43.6 ± 6.2** 8.4 ± 2.125 Gy 4 45.0 ± 6.9 45.1 ± 6.4 9.8 ± 2.730 Gy 7 39.4 ± 3.6** 50.4 ± 3.4** 10.1 ± 2.735 Gy 7 40.5 ± 7.3 50.0 ± 5.9 9.5 ± 2.345 Gy 3 19.5 ± 9.2* 76.5 ± 9.2* 4.0 ± 0*55 Gy 3 10.5 ± 14.8* 84.0 ± 18.4* 5.5 ± 3.5

†Data are expressed as mean ± SD. For comparisons between groups, the Mann-Whitney U test was used. The 20-Gy group was compared with the ‘‘No treatment’’group, and each of the other treatment groups was compared with the 20-Gy group.*P < 0.05.**P < 0.01.

Fractionated Radiation of Dunning R3327-PAP 21

sponse, as seen in both the tumor volume growthcurve and the morphological analysis. With bilateraltumors (a 45-Gy irradiation dose to the tumor on oneside and 55 Gy to the other), the total irradiation doserelated to body surface was too great for these ani-mals, and 7 of 10 rats were lost during the first 4 weeksof follow-up.

The Dunning R3327-PAP tumor normally has agrowth arrest after castration [14], and it is interestingto note that the primarily irradiated, relapsed tumorsin this study responded with a slowing-down or ces-sation of growth after late orchiectomy. This suggeststhat the regrowing tumors after suboptimal doses ofirradiation still contained hormone-sensitive cells. It isnot known whether these suboptimal irradiationdoses primarily kill the more hormone-refractory tu-mor cell populations, leaving the hormone-sensitivecells in a viable condition. There is a growing body ofevidence suggesting that intrinsic characteristics de-riving from the cell type irradiated are important indetermining radiosensitivity [20]. Previous studiescomparing irradiation sensitivity of the poorly differ-entiated Dunning R3327-AT tumor with the hormone-sensitive R3327-H showed that the R3327-AT tumor ismuch more resistant to irradiation, possibly due to ahigher degree of hypoxic areas in the R3327-AT tumor[17]. Studies by Rao et al. [16] showed that the rela-tively high radioresistance in the R3327-AT tumorcould not be fully explained by the presence of hyp-oxic cells, since this tumor also has a high intrinsicradioresistance. The R3327-AT tumor is also more ra-dioresistant than its metastasizing variant, the R3327-MATLyLu tumor, which metastasizes to lymph nodesand lungs, and this was thought to depend on differ-ent intrinsic radioresistances in different tumor stemcells. Concerning the R3327-PAP tumor, there mightbe a difference between androgen-dependent and -in-dependent cells in DNA repair ability immediately af-ter irradiation and a difference in intrinsic radiosensi-tivity.

CONCLUSIONS

This study suggested that fractionated irradiationof androgen-sensitive rat prostatic R3327-PAP adeno-carcinoma induced a dose-dependent delay of tumorgrowth in hormonally intact rats, but no radiationdose chosen eradicated all adenocarcinoma cells. Re-lapsing, primarily irradiated tumors were still andro-gen-sensitive, since orchiectomy reduced the velocityof tumor regrowth.

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