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The growth of transplanted tumours in mice after chronic inhalation of fresh cigarette smoke.

The subcutaneous growth of the Lewis lung tumour in C57BL mice chronically exposed to fresh cigarette smoke was increased above that in age-matched control mice. When murine sarcoma virus (Harvey) induced tumour cells were introduced to the lungs of groups of BALB/c mice, only mice chronically exposed to fresh cigarette smoke died with tumour cells in the lungs. Tumour cell growth in mice during short term cigarette smoke exposure was indistinguishable from that in controls.

CIGARETTE smoking has been associated with an increased incidence of neoplasia in the respiratory system and elsewhere in the body (Royal College of Physicians, 1971;U.S. Public Health Service, 1971). Studies of the influence of cigarette smoke inhalation on pulmonary tumourigenesis in mice exhibiting a relatively high natural incidence of lung adenomata and adenocarcinomata showed an earlier onset and increased incidence of these neoplasms (Leuchtenberger and Leuchtenberger, 1970). However, when a strain of mice with a low natural incidence of these neoplasms was examined, no increase in neoplasms was found (Leuchtenberger, Leuchtenberger and Rossier, 1973). Aqueous extracts of cigarette smoke condensate fed to rats have been found to increase the incidence of tumour production by chemical carcinogens (Sydnor, Allen and Higgens, 1972). The cigarette smoke product(s) could promote carcinogenesis by acting directly on malignant or premalignant cells, or by modifying some regulatory influence of the body opposing the growth of the neoplasm.
We now report the ability of transplantable tumours to grow in mice after short term or chronic cigarette smoke inhalation. The ability of tumours to grow subcutaneously and metastasize to the lungs, as well as the growth of cells introduced directly into the pulmonary system, have been examined.

MATERIALS AND METHODS
Cigarette smoke inhalation.-Strictly line bred BALB/c and C57BL mice were exposed for 7-8 min on weekdays to fresh cigarette smoke in a Hamburg II small animal smoking machine set to give a 1: 7, smoke: air ratio as previously described (Thomas, Holt and Keast, 1973a). The cigarette smoke exposures were continued throughout the duration of all experiments.
Tumours and inoculation procedures.-The Lewis lung tumour of C57BL mice was obtained from Dr Hellman, Imperial Cancer Research Fund, and maintained in our laboratories by serial passage through adult mice. This tumour originated as a spontaneous anaplastic lung carcinoma (Sugiura and Stock, 1955). Preliminary studies showed that the subcutaneous inoculation of 106 viable cells could produce tumours in 18/20 mice. This number of cells was used as the standard inoculum in all experiments. The diameter of the tumours was determined by 2 measurements taken at rightangles to each other, employing calipers. The cube of the mean of the diameters determined by this method and the weight of the tumours were found to have a product moment correlation coefficient of 0X89.
The number of metastases in the lung was determined by the method of Wexler (1966); the lungs were infused with dilute India ink through the trachea, washed, fixed and bleached. The metastases appeared as white nodules on a black background. In order to allow the tumour maximum opportunity to metastasize, the tumour bearing animals were left until a number died or became moribund (about 30 days). All the mice in the test and control groups were then examined for lung secondaries and the significance of the difference between the number of metastases in the groups determined by Student's " t " test.
The TKL5 tumour cells were a clone from a line (W47-A) of tumour cells cultivated in vitro from a tumour induced in a BALB/c mouse by murine sarcoma virus (Harvey) (MSV-H) (Thomas et al., 1973). They were maintained in tissue culture and produced virus. These tumour cells were introduced directly into the respiratory tract. Mice w%vere anaesthetized with pentobarbitone sodium (Nembutal, Abbott Laboratories, Australia) and a blunt 19 gauge needle inserted under the epiglottis and almost to the bottom of the trachea where 105 cells in 0-01 ml of media were introduced.

RESULTS
C57BL mice were exposed to fresh cigarette smoke for 3 days, 23 weeks and 38 weeks before the subcutaneous inoculation of 106 Lewis lung tumour cells.
Cigarette smoke exposure was continued during tumour growth. The growth of the tumours in mice exposed to cigarette smoke for 3 days before the inoculation was almost identical with the growth of tumours in age-matched control mice ( Fig.). However, the growth of the tumours in mice that had been exposed to fresh cigarette smoke for 23 and 38 weeks was significantly increased compared with control animals. The mice examined for metastases in the 2 smoke exposed groups had 2-8 i 1 1 (mean + s.e.) metastases per lung. The corresponding agematched control mice had 0-8 + 0 4 metastases per lung. The growth of Lewis lung tumour in mice exposed to cigarette smoke for 3 days, 23 weeks and 38 weeks. Each result is the mean±s.e. of the tumour diameter of 10 mice: * smokeexposed, 0 control. The significance of the differences between tumour diameter in the cigarette smoke exposed and control mice was determined by Student's " t " test. There was no significant difference between the diameter of the tumour in mice exposed to cigarette smoke for 3 days and the controls. The diameter of the tumours in mice exposed to cigarette smoke for 23 weeks and 38 weeks became significantly different (P < 0 05) from the corresponding control mice on Days 20 and 9 respectively.

THE GROWTH OF TRANSPLANTED TUMOURS IN MICE
BALB/c mice were expose cigarette smoke for 20 weeks introduction of 105 TKL5 cell trachea. As shown in the Tabl 6/13 of the smoke exposed mi became moribund. None of matched control mice died with In another experimeint (Table, group of BALB/c mice were fresh cigarette smoke for 31 w( receiving the intratracheal ino TKL5 cells. As well as an ag control group, this experimen a group of mice of the same control mice but exposed tc smoke for 3 days before the i Only the mice exposed to fres smoke for 31 weeks died or be( bund (6/15). The lungs of were examined histologically. of all the mice examined numerous foci of neoplastic pr These foci varied in size from to large extensive zones of growth which completely rel whole lobes of the lung. however, they were small, co 50-200 cells and were situ bronchi or small vessels. The spindly or irregular in shape, irregular nuclei and basophilic Mitoses were present. In ca foci were large, vasoproliferati evident together with an in lymphocytes, macrophages and In addition, patches of broncho and pulmonary collapse were d to fresh before the Is into the found, which probably contributed to the death of the animals.

DISCUSSION ice died or
The results show that the subcutaneous . the agegrowth of the transplantable Lewis lung n 50 days. tumour in mice was increased by chronic cigarette smoke inhalation. The mice rette Smoke chronically exposed to cigarette smoke ntratracheal had an increased number of metastases in Cells the lung but this was not statistically significant. However, MSV-H tumour Meas t (rdeah) cells introduced directly to the lungs were 28.6 (14-40) able to persist and develop only in the lungs of mice chronically exposed to 15 0 (1A46) cigarette smoke. Other studies in our laboratories (unpublished) suggest that this is not caused by a decrease in tracheoid. bronchial clearance. Short term cigarette smoke inhalation did not produce these effects. It has been shown that handling Expt 2) a stress during cigarette smoke inhalation exposed to experiments does not affect body weight eeks before (Leuchtenberger and Leuchtenberger, iculation of 1970) or immune responses of mice (Esber ge-matched et al., 1973;Thomas et al., 1974). This it included concurs with these results showing that age as the chronic, rather than short term inhalation ) cigarette of cigarette smoke was required to enhance noculation. tumour growth. ,h cigarette While it is now well established that came mori-tobacco contains many carcinogenic subthese mice stances (United States Public Health The lungs Service, 1971), the present experiments exhibited indicate that cigarette smoke inhalation oliferation. was capable of modifying conditions a few cells within the smoking animal that result in neoplastic promotion of growth of an established placed the malignant cell. The fact that conditions Generally, for the production of enhanced tumour insisting of growth do not occur after short term ated near smoke exposure (3 days) indicates that cells were either an accumulation of substances with large acting directly on the tumour cells must cytoplasm. occur, or that the cigarette smoke iiihalaises where tion eventually impairs a mechanism on became controlling tumour growth. We have ifiltrate of previously shown that the immune system giant cells. of mice is impaired by chronic cigarette pneumonia smoke inhalation (Thomas et al., 1973a, b;commonly 1974).
The immune system is thought to exert control on the growth of malignant cells (Burnet, 1970;Keast, 1970). As immunostimulants are known to inhibit the growth of the Lewis lung tumour (Renoux and Renoux, 1972), and the tumour cells produced by MSV are very antigenic (Fefer, McCoy and Glynn, 1967;Law, Ting and Stanton, 1968), it is possible that the cigarette smoke inhalation depressed the ability of the mice to elicit an immune response against the tumour cells. Not all types of neoplasia have been associated with cigarette smoking. However, the effect of immunosuppression on the expression of neoplasia would depend on the amount of control normally exerted by the immune system on that type of neoplasm and the amount of immune control required to prevent tumour growth. This work was supported by the Australian Tobacco Research Foundation. WRT is in receipt of a C.S.I.R.O. postgraduate studentship.