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Effect of age and sex on lung-colony-forming efficiency of injected mouse tumour cells.

The i.v. injection of a specified number of cells of either an Ehrlich ascites tumour (ELD) or spontaneous mouse mammary adenocarcinomas (MA) into C3H mice yielded a number of lung colonies which varied significantly with the age or sex of recipient mice. The yield was higher in mice of 71 weeks than in those of 15 weeks, except for MA cells injected into females, when the yield was higher in the younger mice. Sex did not influence very significantly the yield of colonies from ELD cells; in the case of MA cells the direction of sex differences depended on age. A difference in the effect of pre-immunization with age was not observed.

IF TUMOUR CELLS are injected into the tail veins of rats or mice, tumour colonies will often develop in the lungs, and many investigators have used this technique to study the influence on metastasis of trauma, blood coagulability etc. (Fisher and Fisher, 1967;Wood, Holyoke and Yardley, 1957;Agostino, Grossi and Clifton, 1961;Van Putten et al., 1975). The tumours used in the various investigations have had different lung colony growth characteristics. For example Baserga et al. (1960) found a linear relationship between number of cells injected and number of lung colonies formed, with up to 6 x 105 Ehrlich ascites cells injected. Above this number the relationship became exponential-like. Van den Brenk et al. (1973) observed a non-linear relationship with 2 allogeneic rat tumours, whereas Williams and Till (1966), and Hill and Bush (1969) both obtained linear relationships with polyomatransformed rat embryo cells and a mouse sarcoma (KHT), respectively.
The colony-forming efficiency (CFE) of tumour cells in the lung has been comparatively low for all tumours so far investigated, and there appears to be no significant difference between the clonogenic potential of allogeneic and syngeneic tumour cells (Thompson, 1974a). The CFE of tumour cells can be altered by various procedures: for example, thoracic irradiation prior to injection can lead to increased CFE (Brown, 1973;Thompson, 1974b;Van den Brenk and Kelly, 1974;Withers and Milas, 1973) as can pretreatment with-drugs (Van Putten et al., 1975).
The present investigation examines the effects of age and sex of mice on the CFE of injected tumour cells.

MATERIALS AND METHODS
The tumours used in this investigation were an allogeneic Ehrlich Landschutz Diploid (ELD) tumour and spontaneous C3H mammary adenocarcinomas (MA). The ELD cell line was originally obtained from the Institute of Tumour Biology, Stockholm, in 1962. The cells grow as ascites tumours, and have been maintained either in vivo or in vitro. MA tumours arose spontaneously in breeding female mice of the C3H/Bts strain As ELD cells grow as ascites tumours, it was relatively easy to obtain a suspension of single cells for injection. However, cells of MA tumours had to be released from the solid tumours by treatment with trypsin. It has previously been found that suspensions of single cells of MA tumours do not give rise to colonies in the lungs, and suspensions of cell aggregates were therefore injected, as a linear relationship has been demonstrated between number of aggregates injected and number of lung colonies formed (Thompson, 1974a). Cell aggregates were obtained by passing trypsinized suspensions through graded nylon meshes. Both ELD and MA cells were suspended in Eagle's medium plus 10% foetal calf serum. C3H mice were injected with ELD or MA cells via their tail veins whilst being restrained in Perspex containers. Mice were kept 8 to a cage, and were allowed food and water ad libitum. The animals were selected so that they weighed 30 to 35 g and there was no difference (P > 0 05) between the mean weights of compared groups. There were 8 to 10 mice per experimental group unless otherwise stated. Animals were killed at either 14 days (ELD) or 42 days (MA) post-injection and their lungs were examined for tumour colonies after 300e 200-0 C) io 100-their inflation and subsequent fixation with Bouin's fluid (Thompson, 1974a).
Because a different spontaneous MA was used for each experiment, inter-experimental comparison was not possible.
The immune capacity of 15-week-old and 71-week-old mice, with respect to the 2 tumours, was tested by pre-treatment with lethally irradiated (LI) cells. 1 x 106 LI cells were injected i.p. at 5 weeks and at 1 week prior to the i.v. injection of viable cells. LI cells were irradiated with 10 krad 200 kV X-rays.
Results from different experimental groups were compared using Student's t tests.  into male mice and the resultant number of lung colonies. It can be seen that the shape of the relationship between number of cells injected and number of lung colonies differs for the 2 age groups.
The CFE of ELD cells in male and female mice was also compared, and results are presented in Table I. The difference between males and females was significantly different (P< 0.05) for 71week-old animals, cells injected into males having a higher CFE. There was, however, no significant difference between male and female 15-week-old recipients. 18 51 3 0 62-9± 6-5 115 -4±10-3 61-3± 8-3 65-6± 7-9 190 0±17 -8 (24 micqper group) were found to have a higher CFE than cells injected into 15-week-old male recipients (P < 0.05). With female mice, the situation was reversed, and the 15-week-old recipients had significantly higher colony counts (P < 0.05). Fifteen-week-old males had similar colony counts to 71-week-old females (P = 0.5) although the 71-weekold males had higher counts than the 15week-old females.
Results from animals pretreated with lethally irradiated cells are presented in

DISCUSSION
The attachment and growth of disseminated tumour cells is affected by many different factors. As has been observed, the CFE of tumour cells in the lung may vary considerably with age and sex of the recipients. Possible reasons for the enhanced growth seen in older animals are the immunological and/or hormonal state of the host; and these factors cannot entirely be ruled out, although it does not seem that they are of primary importance. Warren and Vales (1972) have proposed that tumour cells will only develop into metastases after attachment to damaged endothelium, which is likely to be more prevalent in older animals. There is also the possibility that increased rigidity and narrowed lumen of blood vessels in older animals might lead to increased retention of tumour cells. The changes seen in the lungs of older mice are similar to those seen after irradiation, e.g. fibrosis, degeneration of blood vessels (Casarett, 1964), and thus there may be some similarity with the increased CFE of tumour cells seen in irradiated animals 5-68 (Brown, 1973;Thompson, 1974b;Van den Brenk and Kelly, 1974;Withers and Milas, 1973). The age-and sex-dependent changes in lung pathology are being examined at present in animals comparable with those used in this investigation. Preliminary qualitative observations have indicated no gross differences in the histopathology of male and female mice, although specific staining techniques reveal a probable increase in the amount of collagen in 71-week-old animals compared to 15-week-old animals. There are more areas of congestion, emphysema and atelectasis in the older animals.
It is possible that both tumours used in this investigation exhibit a type of cellinteraction growth-enhancement effect. This is demonstrated by the exponentiallike relationship existing between numbers of ELD cells injected and lung colony formation in 71-week-old mice ( Fig.). However, this effect was not observed when cells were injected into 15-week-old mice, although it might become apparent if higher numbers of cells were to be injected. With the MA tumour it is necessary for tumour cells to be in physical contact with each other in order for colony growth to occur. It has been proposed by Willis (1952) that metastases may develop from small clumps of tumour cells rather than from single cells. If this is so, the ability of single-cell suspensions of ELD tumours, and many other transplanted tumours to grow in the lung after i.v. injection may not be analogous to the spontaneous situation. It has been previously demonstrated that, after several transplantations of a spontaneous tumour, single-cell suspensions will grow into lung colonies after i.v. injection (Thompson, 1974a).
The finding that MA tumour cells injected into the older mice had a comparatively low CFE is difficult to explain. The most obvious reason is that the sex hormone status of the host is affecting the CFE of cells. Seventy-one week-old female mice had the same length of oestrus as 15- week-old females, although there could still be a quantitative difference in hormone levels. This remains to be investigated.
Finally, in view of the differences in CFE observed when cells are injected into animals of varying age and sex, it is necessary to emphasize the need for strict standardization when using the lung colony assay technique.