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Annals of Surgical Oncology 9:292-297 (2002)
© 2002 Society of Surgical Oncology

ORIGINAL ARTICLES

An Experimental Evaluation of Three Preoperative Radiation Regimens for Resectable Rectal Cancer

Genc Basha, MD, PhD, Willy Landuyt, SrSc, Jack Fowler, PhD, Dirk Vordermark, MD, PhD, Karin Haustermans, MD, PhD, Karel Geboes, MD, PhD, Walter Van den Bogaert, MD, PhD, Sing Hiem Yap, MD, PhD, Philippe Lambin, MD, PhD and Freddy Penninckx, MD, PhD

From the Departments of Abdominal Surgery (GB, FP), Radiobiology/Radiotherapy (WL, JF, KH, WVdB, PL), Pathology (KG), and Hepatology (SHY), University Hospital Gasthuisberg, Katholieke Universiteit Leuven, Belgium; and Department of Experimental Therapy (DV), The Netherlands Cancer Institute, Amsterdam, The Netherlands.

Correspondence: Address correspondence and reprint requests to: Freddy Penninckx, MD, PhD, Department of Abdominal Surgery, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium; Fax: 32-1-634-4832; E-mail: freddy.penninckx{at}uz.kuleuven.ac.be


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: We investigated the degree of tumor cell killing after radiotherapy regimens commonly used in clinical practice in comparison with an accelerated schedule.

Methods: Mtln3 mammary adenocarcinoma tumor cells were inoculated subcutaneously in the hind leg of syngeneic Fischer 344 rats. Tumors were irradiated with 5 x 5 Gy in 5 days, 10 x 3 Gy over 10 days, or 5 x (2 x 3) Gy in 5 days. After excision of the irradiated tumors, the dye exclusion, a tetrazolium-based colorimetric and the clonogenic assays were used to determine tumor cell viability and surviving fractions.

Results: Estimated potential doubling time values indicate a rapid proliferation capacity, comparable with potential doubling time values in human rectal cancer. The dye exclusion and clonogenic assays revealed a significantly higher degree of cell killing after the hypofractionated and the accelerated regimens of, respectively, 5 x 5 Gy and 5 x (2 x 3) Gy over 5 days compared with 10 x 3 Gy over 10 days.

Conclusions: A shorter treatment time offered the best therapeutic efficacy. The schedule involving two daily fractions of 3 Gy over 5 days should be less toxic than 5 x 5 Gy and may therefore provide a therapeutic advantage.

Key Words: Rectal carcinoma • Preoperative irradiation • Total treatment time • Fractionation • Tumor cell proliferation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In addition to optimized surgery, including total mesorectal excision, adjuvant radiotherapy given before and/or after surgery has been introduced and has been reported to significantly reduce local recurrence rates after curative resection for rectal cancer.1–7 The sequencing of adjuvant therapy for rectal cancer is changing, with greater emphasis on preoperative radiation or chemoradiation. The recent Swedish Rectal Cancer Trial (SRCT), favoring hypofractionated 25-Gy (5 x 5 Gy in 5 days) preoperative radiotherapy, recorded a reduced local recurrence as well as a significant survival advantage after conventional curative resection as compared with postoperative radiotherapy or surgery alone.7,8 Also, standardized total mesorectal excision after preoperative radiotherapy with 25 Gy given in 5 days was found to improve local control in stage II and III mid and lower rectum cancer (C. J. Van de Velde, unpublished data, 2001). However, such an irradiation regimen was found to be associated with impaired long-term bowel function and increased morbidity.9,10

Preoperative radiotherapy in a total dose of 30 to 45 Gy delivered in fractions of 1.8 to 3 Gy/day is mainly used to achieve tumor downstaging and to facilitate curative resection after a 6- to 8-week delay for tumor reduction, particularly for patients with resectable T3 to T4 rectal cancer.11–13 The downstaging effect of the short course of preoperative radiotherapy (5 x 5 Gy given in 5 days) remains controversial and seems to be achieved only if the interval between the start of radiotherapy and surgery exceeds 10 days.11,14 However, experimental and clinical data have demonstrated that clonogenic tumor cells that survive initial irradiation proliferate rapidly during the prolonged fractionation, resulting in failure to control the tumor.15–17 Consequently, a reduction of the overall treatment time has been suggested, to improve the effectiveness of radiotherapy and to increase the chance for cure.18

To our knowledge, no experimental studies have been published comparing the effects of different radiotherapy regimens commonly used in clinical practice. In the search for the optimal preoperative radiotherapy for patients with rectal cancer, we investigated the degree of tumor cell kill by using in vitro assays that followed a simulated standard regimen, a hypofractionated regimen, and an accelerated irradiation scheme. This aims to examine whether a shorter treatment time combined with reduced fraction size, while a relatively high total dose of radiotherapy is maintained, would provide the best therapeutic efficacy.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental Model
Tumor cells used were from the Mtln3 clone derived from the mammary adenocarcinoma cell line 13762NF/Mtln3 developed in the Fischer 344 rat. They were grown and prepared as described previously.19,20 After the cell density was determined, dilutions were prepared with an adjusted number of 107 tumor cells in 300 µl and were preserved on ice until used for injection. Female Fischer 344 rats, 12 weeks old, weighing from 150 to 192 g, were used throughout. Under ether anesthesia, the left hind leg was shaved, and 107 tumor cells in 300 µl of F10/Dulbecco’s modified Eagle medium (DMEM) were injected subcutaneously (SC). Tumor growth was followed up; the length, width, and height of the tumor were measured regularly; and volumes were determined by using an ellipsoid approximation: length x width x height x {pi}/6.21 Animals having tumors with volumes of 1 ml were stratified into three groups before any further manipulation. Each group was treated with a different regimen of radiotherapy and included sham-treated animals. All animal experimental work was performed according to the University Ethical Committee and European Guidelines for animal experiments.

Potential doubling time (Tpot) measurements involved 12 sham-treated animals that received an intraperitoneal injection of iododeoxyuridine (100 mg/kg) 4 hours before tumor excision. After excision, two pieces of tumor were taken from the center and the periphery, fixed in ethanol 70%, stored at 4°C in the dark, and used for Tpot measurements. The Tpot represents the time in which a cell population would double if there were no cell loss and is the inverse of the cell birth rate. The technique has been described previously.22,23

Irradiations
The rats were anesthetized with pentobarbital 6 mg/100 g body weight to allow reproducible prone positioning in the treatment field. Irradiations were performed with a linear accelerator (CGR, Paris, France) by using a beam of 18-MV photons. The dose rate was 200 cGy/min at a focus-to-skin distance of 127 cm. The hind leg with the SC implanted tumor was irradiated while the remainder of the rat was shielded with an 8-cm-thick Arplay Cerro (Arplay, Izeure, France), positioned on top of a 2.5-cm PlexiglasTM (Rohm and Haas company, Philadelphia, PA) plate necessary for electronic equilibrium. Dosimetry in the treatment position was performed by using a rat with LiF pastilles positioned SC at the tumor cell inoculation area; this indicated the accuracy of the dose delivered to the tumor. Three different irradiation schedules were used. The first group received five fractions of 5 Gy each, given over 5 days. A second group received 10 fractions of 3 Gy daily within a total time of 10 days without a weekend gap. The third group was treated with 10 fractions of 3 Gy delivered in 5 days, with 2 fractions a day separated by an interval of 6 hours. Three control animals that received sham treatment under anesthesia were included in all groups. A separate batch of four sham-treated animals did not receive anesthesia, to evaluate its influence on tumor cell viability and proliferation. After irradiation, the influence of the radiotherapy schedule on tumor cell survival was studied.

Tumor Response Assays
Two days after the end of irradiation, under ether anesthesia and in sterile conditions, the tumor was dissected from the surrounding tissue, placed in 30 ml of phosphate-buffered saline, and preserved in ice (4°C). The animals were killed at the end of the surgical procedure with an ether overdose. Next, the phosphate-buffered saline was discarded, and the tumor was placed into a sterile Petri dish. Some 10-ml collagenase solution .05% (Sigma Chemical Co., St. Louis, MO) in buffer II, pH 7.6 [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 24.0 g, NaCl 3.9 g, KCl .5 g, CaCl2·2 H2O .74 g, and distilled water], prewarmed at 37°C, was added to the Petri dish, and the tumor was quickly cleared from necrotic tissue and cut into small pieces. An additional amount of 10-ml collagenase solution was added, and the dish was placed in the incubator at 37°C in an atmosphere of 95% humidified air and 5% CO2 for 15 minutes. After tumor disaggregation, equal volumes of tumor suspension were transferred into centrifuge tubes through nylon cell strainers (FalconTM, Becton Dickinson, Franklin Lakes, NJ) with 100-µm pores. Double volumes of cold minimum essential medium Rega 3 (Gibco, Merelbeke, Belgium) were subsequently added to the tumor cell suspension through the strainers to stop the collagenase activity. Tumor cells were then spun down by centrifugation at 50 x g at 4°C for 6 minutes, and the supernatant was discarded. They were resuspended in 10 ml of Ham’s F10/DMEM to obtain tumor cell suspensions, aliquoted in three tubes at 106 cells per milliliter, and preserved in ice (4°C) for further assessment of tumor cell viability by using the following assays.

Trypan blue assay was used to evaluate cell viability as described previously.24 Stained cells were considered to be no longer viable.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT), a colorimetric assay, was performed by pipetting 300 µl of cell suspension into 96-well microtiter plates; this proceeded as described.25 High optical density readings correspond to a high intensity of dye color, i.e., to a high number of viable cells able to metabolize MTT salts.

Clonogenic assay was performed in standard fashion by plating tumor cells in 25-cm2 flasks to estimate their ability to proliferate and form colonies after irradiation. The aim was to obtain approximately 100 colonies per dish at fixation. Six flasks were plated with 100 and 300 tumor cells each for sham-treated animals. Higher numbers of cells were plated for treated tumors. From tumors treated with 5 x 5 Gy over 5 days, eight flasks were plated with 5 x 103, 1 x 104, 3 x 104, and 1 x 105 cells per dish in a pilot test and 2 x 104, 4 x 104, and 6 x 104 in two other tests. For tumors receiving 2 x 3 Gy every day during 5 days, eight flasks were plated with 1 x 104, 2 x 104, 4 x 104, 5 x 104, and 1 x 105 cells per dish. Finally, for those treated with 3 Gy each day over 10 days, again eight flasks were plated with 1 x 104, 2 x 104, 4 x 104, 5 x 104, 1 x 105, and 2.5 x 105 cells. After adding an appropriate amount of medium (Ham’s F10/DMEM), all flasks were placed in the incubator at 37°C and were processed as described previously.26 The survival values presented in the study are the values of four independent assays for each tumor receiving the corresponding irradiation schedule.

Statistical Analysis
The data are presented as mean ± SEM. Nonparametric tests were used to determine the statistical significance of the difference between surviving values measured with three assays obtained after application of three irradiation schedules. The Mann-Whitney U-test was used for unpaired comparisons. P values (two tailed) smaller than .05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
After SC cell inoculation, tumor take occurred in all animals, and tumors reached the predetermined volumes of 1 ml before irradiation within 2 weeks. Tumor cell kinetic measurements showed characteristics of a rapidly proliferating tumor type. The mean Tpot of 12 separately measured control tumor samples was 2.5 ± .7 days, with a labeling index of .16 ± .04 and a time for the cells to complete the S phase of 9.2 ± 1.5 hours.

The results of the effect of radiotherapy on tumor cell survival are listed in Table 1. As described, a sample from dissociated tumor tissue was analyzed for viability with trypan blue 2 days after irradiation. The ratio of unstained, i.e., viable, cells from both sham and irradiated tumors defined the surviving fraction (Fig. 1A). After sham irradiation, the trypan blue exclusion test revealed an average of 81.7% ± 3% living cells. After all three irradiation schedules, tumor cell viability was significantly reduced as compared with the respective sham-treated tumors (P < .05). Significantly lower percentages of living cells were observed when tumors were irradiated with 5 x 5 Gy or 5 x (2 x 3) Gy delivered in 5 days than in the 10 fractions in 2-week schedules (P < .05).


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  		TABLE 1. The effect of radiotherapy on tumor cell viabilitya
 


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  		FIG. 1. The effect of 3 radiation regimens on Mtln3 cell line–induced tumors as determined by trypan blue assay (A), MTT, a tetrazolium-based colorimetric assay (B), and clonogenic assay (C). The results are shown as mean ± SEM. For sham-treated animals, each data point represents the mean of 3 independent assays for three tumors. The value of the surviving fraction in sham-treated animals was considered to be 1.0. For irradiated tumors, each data point represents the mean of 4 independent assays in triplicate for 4 tumors. Trypan blue assays were performed in triplicate. OD, optical density.

 
The results of the MTT assay were obtained after optimizing both the number of incubated cells and the assay duration time (pilot experiment). Higher readings of absorbance were observed after incubation of the cells derived from the sham-treated tumors (Fig. 1B). After irradiation, a significantly lower amount of formazan was produced in the 5 x 5 Gy and in the 5 x (2 x 3) Gy groups (P < .05). In contrast, when the absorbance values of cells obtained from tumors treated with 10 x 3 Gy over 10 days were compared with the respective sham-treated values, no statistical significance was found (P > .05).

Plating efficiency was calculated for control and treated tumors after seeding an appropriate number of cells to evaluate the surviving fractions after irradiation. The surviving fractions were analyzed separately for irradiated and nonirradiated tumors (Fig. 1C, Table 1). A significantly lower number of cells were found to proliferate and form colonies after irradiation as compared with the sham treatment condition (P < .05). However, after 5 x 5 Gy and 5 x (2 x 3) Gy irradiations delivered in 5 days, lower values of surviving fractions were scored than with the irradiation of 10 x 3 Gy over 10 days (P < .0001).

To compare the three methods used for the assessment of tumor cell viability and proliferation after irradiation, data were normalized with reference (100%) to the results obtained with the respective sham-treated animals. Figure 2 illustrates that the trypan blue assay and the clonogenic assay gave similar degrees of cell killing after the three regimens of radiotherapy. When the MTT assay was used to investigate tumor cell viability, a similar trend (qualitative results) was observed in terms of comparing both 5-day radiotherapy schedules with the 10 x 3 Gy in 10 days regimen.



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  		FIG. 2. Comparison of the 3 methods used to assess tumor cell viability and proliferation after irradiation. Data were normalized with reference to the results obtained in the respective sham-treated animals (these values were put at 100%). The percentage of surviving or colony-forming cells is represented.

 
Cell viability and proliferation of tumors obtained from four sham-treated animals who did not receive anesthesia was also determined by using the previously mentioned assays. Trypan blue assay revealed 89% ± .9% living cells, MTT assay scored .336 ± .041 absorbance values, and the plating efficiency was .69 ± .07. No statistical difference for tumor cell viability and proliferation was observed when compared with cells obtained from the tumors of pentobarbital-anesthetized animals.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although the sequencing of adjuvant radiotherapy for rectal cancer is changing in favor of preoperative radiation, optimization of the regimens remains a matter of concern for both surgeons and radiation oncologists. Therefore, we used an established rodent tumor model to investigate the relative effectiveness of schedules with short and long overall irradiation times as used in several centers. The rat mammary adenocarcinoma Mtln3 cell line used in this study has been previously applied experimentally to investigate recurrent colorectal cancer.20,27,28 It has been discussed that rapid tumor cell proliferation, indicated by a high labeling index and short Tpot (2–5 days), reduces the efficacy of radiotherapy during prolonged treatment times of several weeks.15–17,29 Therefore, the reduction of repopulation of surviving tumor cells during treatment is one of the main factors of success obtained by short radiotherapy schedules.15,17,18,30,31 The preirradiation flow cytometric evaluation indicated a Tpot value of 2.5 ± .7 days, which implies a high in vivo proliferation capacity of the Mtln3 mammary adenocarcinoma. This is very similar with HCT-15 and WiDR human colon carcinoma cell lines, which have Tpot values of 2.2 ± .1 days and 3.3 ± .4 days, respectively.32,33 Furthermore, clinical studies on tumor cell proliferation in colorectal cancer showed a striking similarity with our tumor phenotype. In poorly differentiated colorectal tumors, Tpot was found to be 2.5 days (.7–4.3 days), and in all tumors of the left colon, Tpot was 2.8 days.34 Similar values were estimated from tumor biopsies of patients with primary rectal cancer.35 These findings support the use of the Mtln3 cell line as a relevant tumor model to investigate the effectiveness of radiotherapy for rectal cancer, because the effect in human tumors is expected to be similar, thus allowing the evaluation of the effect of overall treatment time.

We considered in our study the preoperative regimen of 25 Gy delivered in five fractions during 1 week, as proposed and applied in the SRCT.8 We also included a standard regimen that gave 10 daily fractions of 3 Gy for a total of 30 Gy. Finally, we designed a third irradiation regimen that introduced a reduced overall treatment time of 5 days but that again used the total treatment dose of 30 Gy with two fractions of 3 Gy given daily (interval of 6 hours). All regimens were given without a weekend gap. Assuming that the pretreatment Tpot of 2.5 days does not change greatly during irradiation, the 5-day difference between our long schedule and the two short schedules should allow two doublings, i.e., a factor of four greater numbers of cells in the 10-day schedule. This is consistent with observations for the clonogenic and trypan blue assay and less so for the MTT assay.

Short-term viability assays used in our study have been extensively applied to determine the degree of cell kill, to discuss cell kill as a key factor in the onset and rate of repopulation,36 and to compare results of the more laborious but more reliable clonogenic assay. The trypan blue exclusion test is reliable and correlates well with the clonogenic assay, as well as with methods used to determine radiation-induced apoptosis.24,37 In our study, the trypan blue exclusion test and the clonogenic assay yielded very similar results, with the degree of cell kill being significantly greater after both short-term regimens as compared with the standard regimen of 10 days. Recently, MTT assay has also gained much popularity because the method is semiautomated, and large numbers of samples can be analyzed rapidly. After in vitro irradiation of various cell lines, a good correlation has been reported between dye exclusion, MTT, and clonogenic assay.38 However, studies investigating the radiosensitivity of cells from murine solid tumors did not come to the same conclusion.25 Also, in our study, a poor correlation was found between the MTT assay and the two other methods used to determine tumor cell viability after irradiation. This may reflect continued mitochondrial activity associated with MTT reduction and formazan production in radiation-induced dead cells. An alternative explanation may be that immediate regrowth of the cells occurred in vitro, particularly after the 10 x 3 Gy irradiation schedule.

Our data show that the effectiveness of a prolonged irradiation regimen for the neoadjuvant treatment of resectable, fast-proliferating tumors can be questioned. They support the hypofractionated radiation regimen used in the SRCT, with which a reduced rate of local recurrence and an improved survival was observed.8 With respect to this regimen, however, two concerns may be raised. First, the relatively high daily fraction doses of 5 Gy may induce high tumor-bed damage, jeopardizing the recovery of normal tissues. Consequently, this would impair the oxygenation of surviving tumor cells and render them more radioresistant to the subsequent radiation fractions.39 Second, it is well known that large fraction sizes of more than 2 Gy produce unfavorable late sequelae.40 A recently published long-term follow-up report of the SRCT indicates increased morbidity and impaired long-term bowel function.10 To keep the irradiation dose sufficiently high but to aim to achieve safe radiotherapy, we evaluated an accelerated irradiation protocol involving two small doses daily while keeping the overall treatment time less than 1 week. The tumor cell kill after this regimen was comparable with that after 5 x 5 Gy. We assume that when two daily fractions of 3 Gy for 5 days are applied, less tumor bed damage may occur, and therefore tumor oxygenation may be favored.41 This would assist the killing of tumor cells by radiotherapy. In addition, less morbidity can be expected to occur, because standard radiobiological modeling indicates that the risk for late complications from 10 x 3 Gy is at least 25% less than for 5 x 5 Gy. Therefore, the accelerated radiotherapy regimen may provide therapeutic advantages.

These data offer proof of the principle that shorter treatment time combined with reduced fraction size, while a relatively high total dose of radiotherapy is retained, provides the best therapeutic efficacy.

Received for publication July 25, 2001. Accepted for publication December 5, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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