int. 1. Radiation

Oncology

Bid.

Phys.,

1976, Vol.

1, pp. 261-265.

Pergamon

Press.

Printed in the U.S.A.

ENHANCED TUMOR CELL RADIOSENSITIVITY IN ARTIFICIAL PULMONARY METASTASES OF THE LEWIS LUNG CARCINOMA? WILLIAM U. SHIPLEY, M.D.S Department of Biophysics, Institute of Cancer Research and the Royal Marsden Hospital, Sutton, Surrey, England and Department of Radiation Medicine, Harvard Medical School,

Massachusetts General Hospital, Boston, MA 02114, U.S.A.

and JUDITH

A.

STANLEY,

B.Sc. and G. GORDONSTEEL,

Ph.D.

Department of Biophysics, Institute of Cancer Research and The Royal Marsden Hospital, Sutton, Surrey, England TBe~~afnMorsiwoa~nnsponsewasstPdkdintBe~I~cPrclnomP~a or sub&aical ll&astatk tumor model to evaluate the radioeem&ivity of ‘+kroseopie~ depo&s. Tumor eell survival analysis followingin situ *Co y-irr8diation showed that ceils in

1 mm diamttm (04mm’)

were markedly more radkm&fve

pulmonary -

(D. = 106 l-ad, bypoxic fmctim < 0*00!5)than tbot?ein 500 mm’subclltplwoustumors (final 0.36). Thus, unlike the large subcu~us tumom the D,=309md,hypoxkfractb=

were nearly fully perfused wftb oxygen by an in situ hnmor lRUIUd&UlM&T~ mknwascnlature. In addition the oxygenated cells from the smdl tumors were more

INTRODUCTION

Although radiation oncologists have long recognized the importance of tumor size on therapeutic response,‘*4J’t6 relatively few radiobiological studies have evaluated this association.‘*‘4~‘5 Therapeutic success frequently requires that regional “microscopic” or subclinical metastatic deposits be sterilized by wide-field regional radiation at suitably reduced doses such that the patient’s normal tissues are not irreversibly damaged. Such small implants may be sterilized with lower doses than are larger primary tumors merely tThe work contained in this report was partly supported by National Cancer Institute Contract No. NCI-CM-23717 and done while one of us (W.U.S.) was on the Moseley Fellowship from Harvard Medical School, Boston, Massachusetts. SPresent address and address for reprints: Department of Radiation Medicine, Massachusetts General Hospital, Boston, MA 02114, U.S.A.

because fewer cells must be inactivated, however in some situations, it may be that in addition the tumor cells in these small deposits are intrinsically more radiosensitive. This latter possibility formed the basis of the present study. METHODS

AND MATERIALS

Tumor system The Lewis lung carcinoma, a ble epidermoid carcinoma which taneously in the lung of a C57BL used throughout this study.” The

transplantaarose sponmouse was intramuscu-

Acknowledgements-We gratefully acknowledge Dr. 0. C. A. Scott and Prof. M. J. Peckham for helpful advise and criticism throughout the project and Dr. N. M. Blackett for valuable assistance in statistical analysis.

261

262

Radiation Oncology 0 Biology 0 Physics

lar TD50 of our subline was l-3 cells.” Subcutaneous tumors were passaged and grown in 2-3 month old female C57BL mice. As has been described previously,’ for each experiment on subcutaneous tumors, mice bearing tumors were allocated into treated and control groups such that each group contained four tumors and that the mean volumes differed among groups by less than 5%. The mean volume of tumors varied slightly from experiment to experiment; the range of the mean volumes was 450-600 mm’. Pulmonary tumors were prepared as “artificial metastases” by the intravenous injection into isogeneic u&radiated mice of 1 x 10’ viable tumor cells. This technique, developed previously for the lung colony-forming ability assay for these tumor cells, yielded after 13 or 14 days growth about 25 pulmonary tumors per animal with a mean diameter of 1.0 mm and a mean tumor volume of 0.5 mm3.9 In experiments assaying the tumor cell radiosensitivity of pulmonary tumors of merent sizes, the artificial metastases were removed following longer intervals after intravenous transplantation so that their mean diameters were significantly larger than the 13 and 14 day tumors. Pulmonary tumors were excised 15 and 20 days after intravenous transplantation; careful measurement with vernier calipers showed their mean diameters to be 2.2 and 3.5 mm, respectively. Irradiations All irradiations

were given as whole body doses to unanesthetized mice at a 6oCo y-ray dose rate of 350-38orad/min. In vitro y-irradiations were given at 300350rad/min to suspensions of single tumor cells (1 x 1Wcells/ml) suspended in Ham’s F-12 medium (Biocult Laboratories, Glasgow, United Kingdom) in equilibrium with air at 37°C. Preparation

in situ

of cell suspension

Immediately following irradiation subcutaneous tumors were excised and minced into =l mm fragments and a suspension of single cells was prepared by a combined mechanical and enzymatic technique as previously described.’ Immediately following treat-

Vol. 1, No. 3-4

ment of these mice bearing pulmonary tumors (three mice per group), the lungs were removed and 20 pulmonary tumors, carefully selected to be l&1*2 mm in dia., 2.2 mm in dia., or 3.5 mm in dia. were excised from the control and treated groups. These tumors were then minced and a suspension of single cells was prepared by the techniques used for the larger tumors. Cell survival assay In uitm colony-forming ability in soft agar (0.3% Bacto-agar, Difco Laboratories, Detroit, Mich.) was measured by the technique developed for this subline of Lewis lung carcinoma.’ This technique using a double layer of soft agar yielded tight spherical colonies containing an average of more than 500 cells after 10-12 days incubation. Plating efficiencies with this technique ranged from 20 to 50%. The surviving fractions were calculated as the ratio of the cloning efficiencies of the irradiated tumor cells to the untreated tumor cells. The standard errors of the mean are shown for all surviving fractions where larger than the symbol, except when multiple similar results were available for a specific dose level and thus error bars could not be clearly shown. Survival curve characteristics, i.e. Do, 0, and n, and their 95% confidence limits, were computed by linear regression analysis on all results at dose levels yielding surviving fractions 10.10. RESULTS AND DISCUSSION

In air breathing unanesthetized mice the cells from O-5mm3 pulmonary tumors are markedly more sensitive to r-irradiation than those from 500 mm3 subcutaneous tumors (Fig. 1A). The major reason for the difference was the radioresistant terminal portion of the survival curve for cells from the large subcutaneous tumors. This slope (Do = 309 rad) is similar to that for tumor cells made acutely hypoxic before in situ y-irradiation by nitrogen asphyxiation (DO= 307 rad, ref. 9). These large Lewis lung tumors contained a fraction of “chronically” hypoxic cells (i.e. those cells within the tumor that are hypoxic under normal conditions) of O-36.9Thus these hypoxic cells dominated the cell population

263

The Lewis lung carcinoma 0 W. U. SHIPLEYetal.

A

SOLID

10:

8. SINGLE CELL SUSPENSIONS

TUMORS

‘F\

2

I

0.5mm3 PULMONARY TUMORS

t in vitro _-

Fig. 1. Yo y-ray cell survival curves for the Lewis lung carcinoma. 1A; tumors treated in situ in air Fig. 2. @‘Co y-ray survival curves of cells from breathing, unanestbetized mice. 1B; well oxyge0.5 mm’ pulmonary tumors treated in vitro as well nated cell suspensions irradiated in vitro. Symbols: oxygenated suspensions (A) or in situ in air 0, A, cells from 0.5 mm3 pulmonary tumors; 0, ?? , breathing mice (0). cells from 500 mm3 subcutaneous tumors.

these cells dose of y-rays due to their radioresistance relative to well oxygenated cells. The survival curve characteristics of cells from O-5mm3 pulmonary tumors irradiation in situ in air breathing mice were similar to those of the well oxygenated cell suspensions irradiated in vitro (Table 1, Fig. 2). Thus the hypoxic fraction of the O-5mm pulmonary tumors was less than O-005 because no resistant tail on the curve was observed over 3.5 decades of survival for surviving

a large single

Table 1. Yo

when breathing mice. A histological subcutaneous and

irradiated in situ in air

comparison of the large small pulmonary tumors supported the radiobiologicallydetermined diflerences in their fractions of chronically hypoxic cells. On microscopic evaluation of the 5OOmm’subcutaneous tumors contained numerous areas of tumor cell necrosis whereas the 1 mm diameter pulmonary tumors had no evidence of necrosis; centrally, they contained one or more capillaries, often

y-ray survival curve characteristics of Lewis lung carcinoma cells

CONDITIONS OF IRRADIATION

500mm' Subcutaneous

D,, rad -

n

Tumors:

in vitro, single cells

111 (99-127)+

in situ, air breathing --

309 (271-360)

0.5mm3 Pulmonary

Dq rad -

342

22 (?I-60)+

Tumors:

in vitro, single cells

89 (63-148)

267

20 (.5-900)

in situ, air breathing --

106 (72-198)

318

20 (.4-1100)

+95% confidencelimits in parentheses

264

Radiation Oncology 0 Biology 0 Physics

dilated, that were filled with erythrocytes. In some histological sections, the 3.5 mm diameter tumors did show small areas of necrosis. Two previous studies have reported increases in hypoxic fraction with increase in tumor size for two solid experimental tumors that were not growing in the 1ung.8*‘3Thus the low (undetectable) hypoxic fraction in the O-5 mm3 pulmonary metastases of the Lewis lung carcinoma is probably a result of their small size rather than their site of growth. Supporting this conclusion are our preliminary observations, summarized in Table 2 from four experiments on the in situ radiosensitivity of tumor cells from artificial pulmonary metastases of mean diameters of 2.2 and 3.5 mm. The @Co y-ray in situ cell survival curves from these larger pulmonary tumors had a terminal portion with a high Do (~27Orad) characteristic of a small hypoxic subpopulation. The fraction of hypoxic cells, calculated as the vertical displacement of the survival curves of cells from pulmonary tumors of air-breathing and nitrogen-asphyxiated mice at 1% survival,9*‘o was 0.01 for 2.2 mm diameter tumors and 0.04 for 3.5 mm diameter tumors (Table 2). Thus for tumors originating within the same tissue we observed, above 1 mm diameter, an increase in hypoxic fraction with an increase in size of these artificial pulmonary metastases. Durand and Sutherland, who studied V-79 Chinese hamster cells grown in vitro as “spheroids’*, reported that intercellular contact increased the shoulder of the survival curve.3 There studies suggested that, because of intercellular contact, tumor cells in situ might accumulate and repair considerably more sublethal radiation damage than predicted by studies on tumor cell lines grown in tissue culture. However, in our studies we

Vol. 1, No. 3-4

found no influence of in situ intercellular contact on the shape of the y-ray survival curve. Cells from O-5mm pulmonary tumors irradiated in situ and in oitro had similar Do and n values (Table 1). The Do for cells irradiated as in vitro suspensions was 20% less for cells from the pulmonary tumors (Do = 89 rad) than for those from subcutaneous tumors (Do = 111 rad). While this difference in radiosensitivity seems real, the scatter in the results for pulmonary tumor cells does not make this a statistically significant result (Fig. 1B and Table 1). The reason for this probable increase in radiosensitivity is not clear. Differences in the age density distribution of these two asynchronous populations could explain this result. However evaluation of this possibility must await completion of our cell population kinetics studies on these two types of Lewis lung carcinoma. The four findings of this study were (1) that 1 mm diameter tumors were fully perfused in situ by oxygen, (2) that, above 1 mm in diameter, a small fraction of hypoxic cells was observed and that this hypoxic fraction increased with an increase in size of the pulmonary tumor, (3) that cells within small pulmonary implants had greater radiosensitivity than those in larger tumors, and (4) that in situ intercellular contact does not increase the shoulder of the radiation survival curve for Lewis lung carcinoma cells. These findings and the similar results reported simultaneously in the EMT-6 tumor system6 may in part explain and predict the success with regional radiotherapy at well tolerated doses in achieving long term local control in subclinical regional deposits of metastatic carcinomas.4J,‘6The similar findings in the EMT-6 and in the Lewis lung tumor systems offer

Table 2. Hvboxic fraction vs tumor size in Lewis hmg carcinoma TIXJR DIAMETER

TUMOR VOLW

1.0 ml

0.5 mm3

2.2 mm

6.0 imn3

3.5 mm 10

mm

20.0 mm3 500

mm3

HYPOXIC

llMDR

FRACTION

SITE