It has been suggested that local factors at the site of growth of a primary tumor might influence the outcome of the metastatic process. Compilation of the data from the literature revealed that growth of tumor cells in the selective medium of the intraperitoneal cavity, of the lymph node and/or of the spleen leads to progression towards a population of cells with a higher metastatic capacity. In search for an experimental model with transplantable rodent tumors that could be used to study the influence of the anatomic site of an implant on the formation of spontaneous metastases, we have considered heterogeneity of microenvironmental conditions in the subcutaneous milieu. For the MO4 mouse fibrosarcoma, a primary tumor growing subcutaneously in the tail was highly metastatic to lymph nodes and lungs while it failed to produce metastases when growing in the pinna. Implantation of a spheroidal aggregate of MO4 tumor cells, alternatively in the tail and in the pinna of syngeneic C3H/He mice, might be an appropriate model, which is discussed in this review.

Abstract. A gnobiotic piglet, was inoculated intracerebrally with hemagglutinating encephalomyelitis virus (strain VWS72). Mononuclear cells formed vascular cuffs and were disseminated in the brain parenchyma. A few neurons were surrounded by the same kind of cells. Virus particles morphologically similar to coronavirus particles were found in the cytoplasm of both chromatolytic light neurons and hyperchromic dark neurons. The particles were in vesicles of distended endoplasmic reticulum and in the hypertrophied Golgi apparatus. Although the morphogenesis of vomiting and wasting disease virus and its effect on cell cultures have been studied ultrastructurally 12, 131, in vivo research has not been reported. Our study shows viral replication in the nervous tissue and ultrastructural cell changes caused by this virus. Materials and MethodsThe virus was obtained from the 10th passage in primary pig kidney cells of a strain (VWS72) originally isolated I IS] from the tonsils o f a naturally infected pig. The biological and physicochemical characteristics of this strain have been reported I IS].A colostrum-deprived 4-day-old piglet was infected intracerebrally . Five days after inoculation the animal was anacsthctizcd with hypnodyl (0.2 ml/kg). Fixation was by perfusion through the abdominal aorta with I % glutaraldchydc. 2% formaldehydc buffered at pH 7.4 with a 0.1 mol/l cacodylatc solution. A 10-day-old spccific pathogen free and colostrum deprived pig was a control. I t was anacsthesized and fixed as was the infected pig. The brains were removed from the skull after 2 hours at 4" C and left in fresh aldehyde fixative overnight. Several I-millimeter cubes were then disscctcd from cerebral cortex. hippocampal region. medulla and pons. They were fixed in I % osmium tetroxide and embedded in spurr medium 1201. 102

MO4 cell aggregates with a diameter of 0.3 mm produced invasive fibrosarcomas after s.c. implantation into the pinna of syngeneic mice. Histology of pinnae fixed 10 min to 5 days after implantation of an aggregate suggested that the tumour was produced by the cells that invaded during the first day, and that the cells remaining in the aggregate were eliminated by the reaction of the host. Before implantation we have pretreated MO4 cell aggregates with 1 microgram/ml of the microtubule inhibitors Nocodazole (ND) and vincristine (VCR), known to inhibit both proliferation and invasion, and with 1 microgram/ml 5-fluorouracil (5-FU), known to inhibit proliferation but not invasion. Tumorigenicity was significantly reduced after treatment with ND or VCR, as compared to treatment with 5-FU or to controls. Histology of pinnae fixed 10 min to 3 days after implantation showed absence or scarceness of invasive MO4 cells after pretreatment with ND or VCR, in contrast with controls or with aggregates pretreated with 5-FU. The effect of ND, VCR and 5-FU on the growth of aggregates in culture on gyrotory shaker was reversible within 1 and 2 days respectively. After treatment with ND or VCR slight alterations in the function of the cytoplasmic microtubule complex remained visible during 3 days in cells migrating from an aggregate explanted on glass. Confrontation of pretreated aggregates with fragments of embryonic chick cardiac muscle in three-dimensional culture indicated that the anti-invasive effect of ND or VCR was reversible in vitro. We concluded that a delay of invasiveness caused by pretreatment with ND or VCR provided the host with the opportunity to eliminate MO4 cells implanted s.c. into the pinna.

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