The shedding of viable cells into the local lymph by tumours growing in the gut of rats

Abstract

Summary Suspensions of syngeneic sarcoma cells were injected into the Peyer's patches of rats from which the mesenteric nodes had been removed. By later cannulating the thoracic duct of such rats it was possible to collect peripheral intestinal lymph that had come directly from the tumour bearing area without being filtered through a yregional node. The first step in the metastasis of malignant tumours is likely to be the shedding of cells into the local tissue fluid (lymph) but it is difficult to study this process under anything approaching "physiological" conditions (Weiss et al., 1980). The lymphatic systems of laboratory rodents are too small usually for direct experimentation, and although this difficulty of size can be overcome by using tumour-bearing sheep (Hall et al., 1975) the nature of the available tumours and the lack of inbred animals pose other severe problems.By excising the mesenteric lymph nodes, allowing a period for the regeneration of the lymphatic vessels, and then cannulating the thoracic (or intestinal) duct, it is possible to collect peripheral (i.e. afferent to the erstwhile mesenteric nodes) intestinal lymph (Hall et al., 1977). Also, by injecting suspensions of syngeneic sarcoma cells into the Peyer's patches of the small gut, it is possible to establish tumours which grow for a few weeks in the wall of the gut without causing obstruction or ulceration (Gyure et al., 1980). When these two techniques were combined it became possible to collect rat lymph which came directly from a tumour bearing area and which had not been filtered through a lymph node (Moore et al., 1982;Styles et al., 1984 intestinal tumours in unanaesthetised rats, and the results of this study are reported here. Materials and methodsGeneral procedure Young, male hooded rats weighing 200-250 g were subjected to mesenteric lymphadenectomy; 6-8 weeks later their abdomens were re-opened and suspensions of syngeneic sarcoma cells were injected into each of the 6-8 major Peyer's patches in the small intestine. From 1-20 days thereafter each rat was provided with a cannula in the cysterna chyli and placed in a Bollman cage so that thoracic duct (i.e. mainly intestinal) lymph could be collected quantitatively. Lymph was collected over periods of 24hr under sterile conditions, and the number of viable tumour cells in each collection was determined by culturing the washed lymph cells in a limiting dilution assay.Similar experiments were also carried out on a control group of intact rats, i.e. rats which had not had their mesenteric nodes removed by prior surgery.In order to take into account the effects of the specific immune responses to the tumour, which must be presumed to have developed during the course of the experiment, an identical series of experiments was carried out on athymic (nude) rats.Animals, tumours, surgical procedures Ten week old specific pathogen free Lister hooded/Ola (RTlC) rats were taken from our own colony, which is maintained in positive pressure isolators.

Immunity to a syngeneic sarcoma induced in rats by dendritic lymph cells exposed to the tumour either in vivo or in vitro

“…Young (5-6 weeks old) male hooded rats were subjected to mesenetric lymphadenectomy so that 6-8 weeks later, after they had attained adult weight (200-250 g) (Gyure et al, 1985) A nimnals, tliaIours anId surgical procedures Specific pathogen free CBH/Cbi (RT1') rats were taken from our own colony, which is maintained in positive pressure isolators.…”

“…This can be done in rats bearing intestinal tumours (Moore et al, 1982;Gyure, et al. 1985) and this type of preparation has allowed us to collect dendritic cells coming directly from the area of tumour growtll.…”

“…Each morning the lymph from several donor rats was pooled and the cells centrifuged over a layer of Nycodenz (S.G. 1.065); the dendritic cells and macrophages remained at the interface, from which they were collected by aspiration, after the lymphocytes and other cells had sunk through the Nycodenz and formed a pellet at the bottom of the tube. The cells thus collected were incubated overnight in plastic culture flasks so that conventional macrophages and any tumour cells (Gyure et al, 1985) adhered to the bottom of the flask. By gently decanting the supernatant fluid it was possible to obtain a population of dendtritic cells contaminated by nothing more than a few lymphocytes.…”