??Session II

ENHANCEMENT OF ARTIFICIAL LUNG METASTASES BY MISONIDAZOLE

SARA ROCKWELL, PH.D., MARIANNE NIERENBURG, A.A.S. AND CAROLYN G. IRVIN, M.S.

Department of Therapeutic Radiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 065 10, USA

The effect of treatment with the hypoxic cell radiosensitizer misonidazole on the formation of artificial lung metastases was examined. Both single treatments with large doses of misonidazole and fractionated treatments with smaller doses of misonidazole were found to increase the number of lung tumors developing after intravenous injection of EMT6 mouse mammary carcinoma ceils. The immunologic and physiologic effects of the nitroimidazole were postulated to be responsible for the enhancement of lung tumor formation.

Misonidazole, Metastases, Hypoxic cell radiosensitizers.

INTRODUCI’ION

The development of lung nodules from intravenously injected tumor cells is frequently used to model the en- trapment and growth phases of the formation of lung metastases. Many factors influence the formation of lung tumors from i.v.-injected tumor cells. Host characteristics such as age, sex, and immunologic status, are important in some systems.6 The induction of stress may alter the incidence of metastases, as does treatment with adrenaline or /3-adrenergic agonists. ‘* Treatment of the host with anti-cancer agents may alter metastasis. Prior lung irra- diation increases the number of lung tumors; the size of the increase depends on the radiation dose and the time between irradiation and injection of cells.‘,‘4 Prior therapy with a variety of antineoplastic drugs (including Cyclo- phosphamide, ACT D, Bleo, CCNU, HU, Methotrexate, and 5 FU) also enhances lung nodule formation.2*‘0*‘3 Recently, Milas et ~1.~ demonstrated that treatment with misonidazole (MISO) enhances the development of lung tumors after i.v. injection of cells from 2 C3H fibrosar- comas. The experiments reported here were undertaken to determine whether MIS0 in single and fractionated regimens alters the formation of lung nodules after i.v.- injection of EMT6 mouse mammary tumor cells into BALB/c mice.

METHODS AND MATERIALS

Mice and tumors Female BALB/c KaRw mice 2.5 months of age.were

used. Mice were bred and maintained in a barrier colony at Yale,5.6 were routinely monitored for microbiological

status, and were free of identified pathogens at the time of this study. EMT6 mouse mammary tumor cells, subline EMT6 Rw, were maintained by alternate passage in mice and in cell culture as described previously,’ and were routinely tested and found to be free of contamination with mycoplasma, pathogenic viruses, or microorganisms. The characteristics of EMT6 cells in vitro and EMT6 tumors in mice are detailed elsewhere.5.6

Lung colonies The ability of EMT6 cells to form lung tumors was

assayed as detailed previously.2*3*6 Cell suspensions were prepared from exponentially-growing cultures. Cells were counted and diluted to obtain a single cell suspension containing 2 X lo4 cells/ml. All mice within an experiment were injected in random order with 0.2 ml aliquots from the same suspension; 6-10 animals were used in each group. The lungs were not preirradiated; no heavily-ir- radiated cells or microspheres were admixed with the viable cells. Animals were sacrificed 14 days after injection of the cells, and the lungs were removed, cleaned of ex- traneous tissues, fixed in Bouin’s, washed with 95% ethanol, and stored in ethanol until they were counted. Lungs were coded and counted blind. Tumor nodules on the lung surfaces were counted using a dissecting mi- croscope. Differences between groups were assessed with Student’s t tests and Mann Whitney U tests; as the results were similar, only the results of the t tests are shown.

Tumor growth Tumor growth was followed by serial external caliper

measurements of the three tumor diameters; volumes

Reprint requests to: Sara Rockwell. Acknowledgment-This investigation was supported by Grant CA-06519. The authors thank Dr. D. Lynn Kirkpatrick for presenting this paper at the meeting.

Accepted for publication 22 March 1984.

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I396 Radiation Oncology 0 Biology 0 Physics August 1984, Volume 10. Number 8

were calculated assuming that the tumors were hemiel- Table 2. Lung nodules in untreated control mice and in mice lipsoids.‘q6 given 5 daily treatments with misonidazole or saline

Misonidazole 5 treatments, day -1 to +4

MISO* was dissolved in warm physiologic saline and injected i.p. Mice injected with single doses received 1 mg/g MIS0 24 hrs before or 24 hrs after injection of the tumor cells. Animals receiving fractionated treatments received 5 injections of 0.5 or 0.2 mg/g/day MISO, given 1 day before and 4 hrs, 1, 2, and 3 days after injection of the cells. This schedule was chosen because our previous studies9 suggest that it should produce immunosuppres- sion throughout the period in which iv-injected cells are circulating, seeding, and beginning to grow. The MIS0 treatment on the day of tumor cell injection was given 4 hrs after injection of cells in order to minimize the importance of the acute hemodynamic effects of MISO. Control mice for this regimen received 5 injections of saline at the same times.

0.5 mg/g 0.2 mg/g Control Saline MIS0 MIS0

9.8 + 2.0 5.0 28.5 f 9.3. 18.5 + 5.6t 16.0 + 2.7 10.1 k 2.1 29.7 + 4.9.4 12.9 + 2.4 17.1 + 3.3 9.4 f 2.9 9 17.1 f 2.4$

Note: Values are lung colonies/4000 cells injected (mean + S.E.M.).

??p < .05 relative to control. t .05 -z p < .1 relative to control. $ p < .05 relative to saline-injected control. Q Group lost, water bottle failure.

RESULTS

Table 1 shows data from 4 experiments examining the effect of MIS0 on lung nodule formation. In all exper- iments, a single treatment with 1 ms/g MIS0 1 day before injection of the cells enhanced lung colony formation (Student’s t test, p < .05). MIS0 given one day after injection of cells increased lung colony formation in all 4 experiments, but this increase was smaller than that when MIS0 was given before injection of the cells and was significant in only 1 experiment. For the total data with MIS0 given on day + 1, the increase was marginally significant (paired Student’s t test .05 < p < .iO). There was no difference in the size or appearance of lung nodules in controls and in mice treated with MISO.

Three experiments (Table 2) also examined the effect on lung nodule formation of 5 treatments with MISO, given 1 day before and 4 hrs, 1,2, and 3 days aher injection of the cells. To control for the stress of the repeated treat-

ments, a group injected 5 times with saline was included in each experiment; fewer colonies developed in the stressed, saline-injected animals than in the untreated control. In contrast, 5 injections of 0.5 mg/g/day MIS0 significantly increased the number of lung colonies in both experiments in which this could be tested. Five treat- ments with 0.2 mg/g/day increased the number of tumors over that in saline-injected controls in all 3 experiments, but this increase was smaller than that observed with 0.5 mg/g/day and was statistically significant in only 1 ex- periment. For the total data with 0.2 mg/g/day, a paired t test showed the increase to be marginally significant (.05 < p < .I). There was no difference in the size or appearance of the lung tumors in treated and control animals.

To ascertain whether the enhancement of lung tumor formation by MIS0 reflected a generalized enhancement of tumor growth, the effect of MIS0 on the growth of established intradermal tumors was examined (Fig. 1). Neither 1 treatment with 1 mg/g MIS0 nor up to 10 daily or twice-daily treatments with 0.5 mg/g MIS0 al- tered tumor growth.

DISCUSSION Table I. Lung nodules in controls and mice receiving 1 mg/g

MIS0 1 day before or I day after injection of tumor cells

Misonidazole

Control Day -1 Day +l

5.8 + 1.2 35.0 +- 4.8* 7.2 2 1.2 9.8 k 2.0 19.8 f 4.8+ 12.9 + 1.7

16.0 f 2.7 44.9 2 4.8* 26.4 +- 2.2* 17.1 + 3.3 24.1 2 4.4+ 19.3 + 2.8

Note: Values are lung colonies/4000 cells (mean k S.E.M.). * Significantly different from controi (p < .05).

Single and fractionated treatments with MIS0 en- hanced the formation of lung colonies in mice injected i.v. with tumor cells. Our findings with MIS0 given before irradiation are in agreement with those of Milas et al.,* who showed that MIS0 injected 1 hr to 2 days before cells at doses r 1 mg/g increased the number of lung metastases. Azothioprine, a nitroimidazole used clinically to suppress graft rejection, which is also a hypoxic ra- diosensitizer, enhances lung tumor formation in an os- teosarcoma. I3

Our data do not identify the mechanism(s) of the en-

* Hoffman-LaRoche.

Misonidazole enhances metastasis ??S. ROCKWELL ef al. 1397

500

: 300

3 200

$ a 0 100

0 2 4 6 8 IO 12 14

DAYS

Fig. 1. Effect of MIS0 on tumor growth. 0: controls. 0: 1 X I mg/g, day 0. A: 5 X 0.5 mg/g days 1-5. m: 10 X 0.5 mg./g. 2 injections/day (8 AM, 4 PM), days 1-5. V: 10 X 0.5 mg/g, days l-10. Points: mean volumes, 6 mice/group. The stippled area covers the standard errors of the means of the control tumors. Arrows indicate the times of MIS0 treatment in the different groups.

hancement of lung colony formation. The formation of lung tumors is also enhanced by both radiotherapy’,‘* and chemotherapy. 2.‘o.‘2~‘3 Several mechanisms have ‘been proposed to explain this enhancement, including direct damage to the lung or its vasculature; suppression of anti- tumor immunity; alterations of blood flow; and the in- duction of physiologic stress, resulting in the liberation of adrenalin and other endogenous substances which in turn alter tumor formation. The increase in lung colony formation by MIS0 does not reflect a general stimulation of tumor growth (Fig. 1)’ ’ or a general increase in tumor cell clonogenicity.‘** Milas et aL4 found that enhancement of lung nodule formation by MIS0 and radiation were independent, and that enhancement by MIS0 was not

1.

2.

3.

4.

5.

altered by hyperbaric 02, suggesting that lung hypoxia is not involved.

MIS0 produces a variety of short-term physiologic ef- fects in mice, including alterations of body temperature and heart rate, and induction of anesthesia-like effects.437q8 These acute physiologic effects cannot be directly re- sponsible for the changes in metastatic efficiency, as they resolve within a few hours, while effects on metastasis remain l-2 days later (Table 1).4 Moreover, fractionated treatments with lower doses of MISO, which minimized the acute physiologic and hemodynamic effects of MISO, also enhanced lung nodule formation (Table 2). Single and fractionated MIS0 treatments inhibit delayed hy- persensitivity in mice’ and may influence tumor cure via immunologic mechanisms. ’ ’ The enhanced formation of metastases may be related to this immunosuppression, as it has been established in many experimental systems, including EMT6, that host immunocompetence influ- ences metastasis.4*6 Our finding that MIS0 treatments given after injection of the cells slightly enhance lung tumor formation suggests that factors other than or in addition to changes in the entrapment of circulating cells are involved.

The clinical relevance of the enhancement of lung nod- ule formation by MISO, like the enhancement produced by radiation or chemotherapy, is uncertain. The MIS0 doses used in this study were large and produced peak drug levels higher than those produced by doses tolerated in man. However, the drug half-life is much longer in people than in mice, and the area under the curve of concentration X time is probably greater for clinical reg- imens in man than for these doses in mice.” While this increased contact time may facilitate the use of nitroim- idazoles as cytotoxic, chemotherapeutic agents in man,” it will also increase any toxicities which are related to contact time rather than peak drug levels. Therefore, the possibility that MIS0 could enhance the formation of metastases should be considered when the agent is used to treat patients with good prognosis for local control and long-term survival.

REFERENCES

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for studying the response of tumors to therapy. Lab. Anim. Sci. 27: 831-851, 1977. Rockwell, S.: Effect of host age on the transplantation, growth, and radiation response of EMT6 tumors. Cancer Res. 41: 527-531, 1981. Rockwell, S.: Hypoxic cells as targets for cancer chemo- therapy. In The Development of Target Oriented Anlicancer Drugs, Y.C. Cheng, B. Goz, and M. Minkoff (Eds.). New York. Raven Press. 1983, pp. 157-172. Rockwell, S.: Combination therapv with misonidazole and mitomycin C: Lack of chemosensitization of EMT6 tumor cells in vivo and in vitro. Int. J. Radiat. Oncol. Biol. Phrjs. 10 (In press) 1984.

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