Site-induced differences in spontaneous metastasis of MO4 mouse fibrosarcoma cells.

MO4 mouse fibrosarcoma cells metastasized to the lungs and to the regional lymph nodes from a subcutaneous tumour in the pinna when they were derived from a tumour in the tail or from lung metastases. In contrast, no metastases were found when MO4 cells were derived from a tumour in the pinna or from the parent cell line. Cells from tumours in the pinna produced metastatic tumours when they were implanted in the tail but not in the pinna. The type of implant-spheroidal cell aggregate, freshly cut tumour fragment or precultured tumour fragment- had no influence on the metastatic behaviour of the resulting tumour. These observations confirm the concept that local factors at the growth site of a tumour influence the metastatic behaviour of that tumour.

Invasiveness and tumorigenicity of MO4 mouse fibrosarcoma cells pretreated with microtubule inhibitors.

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.

Influence of implantation site on formation of metastases.

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.

Influence of implantation site of MO4 cell aggregates on the formation of metastases.

Implantation of spheroidal aggregates of virally transformed MO4 fibroblastic cells into the pinna or into the tail of syngeneic C3H mice in matched experiments produced invasive fibrosarcomas in all animals. The growth rate of primary tumours was lower in the tail than in the pinna. Histology revealed no differences between tumours at both sites of implantation. Fibrosarcomas in the tail formed spontaneous metastases in the lungs and in the ischiadic, lumbar, renal and thoracic lymph nodes in 69-85% of the mice. After comparable tumour-bearing periods, no metastases were observed in mice with tumours in the pinna. Our experiments clearly demonstrated the influence of the site of the primary tumour on the formation of metastases.

Invasion of malignant cells in vivo and in vitro: similarities and differences.

Progressive occupation of other tissues, accompanied by degeneration of these tissues indicates tumour invasiveness. Tumour cells presumed to be invading show alterations in shape, produce irregular cytoplasmic extensions, contain microfilament bundles and heterophagosomes. Confrontation of malignant cells with fragments of normal tissues in threedimensional culture produces pictures of invasion that would allow the pathologist to recognize that the tumour cells are malignant. Invasion of cells in these cultures correlates with formation of invasive tumours in syngeneic animals. Confrontation of malignant cells with non-malignant cells or tissues in twodimensional culture on artificial substrate mimics invasion less well. Twodimensional cultures might nevertheless present valuable complements to other models for the study of the activities of invasive cells. It is concluded that in vitro techniques deserve further interest from students of invasiveness because of their contribution to new insights into the mechanisms of invasions.

Invasion of malignant C3H mouse fibroblasts from aggregates transplanted into the auricles of syngenic mice.

Spheroid aggregates of malignant fibroblasts (MO4), shown to be invasive in vitro, were implanted subcutaneously into the auricle of the external ear of syngenic C3H mice. The course of early invasion into the surrounding tissues and the formation of tumours was studied in serial sections of auricles fixed 6 h to 30 days after implantation. MO4 cells are first observed to make contact with the surrounding tissues after 6 h. They exhibit, cytoplasmic extensions and spread from the original aggregate. During the first day invading MO4 cells preferentially follow tissue crevices created by the inoculation procedure. Later they also invade the surrounding tissues. Polymorphonuclear leucocytes, and later monocytes infiltrate the aggregate, which is completely destroyed after 4 days. Palpable tumours arise from MO4 cells that have left the original implant and invaded the tissues of the auricles. These observations indicate that invasion of malignant cells is important for the take of a transplant.

Methods of study of the invasion of malignant C3H-mouse fibroblasts into embryonic chick heart in vitro.

Methods of study of tumour invasiveness in vitro were investigated using the interactions between malignant virally transformed C3H mouse fibroblasts (MO4) with fragments from embryonic chick heart. Invasion of MO4 cells into the heart tissue could be demonstrated in all three-dimensional cultures. On the contrary, seeding of MO4 cells on top of a monolayer of heart cells did not mimic invasion. Precultured heart fragments were more suitable for the study of the early phase of invasion than freshly cut ones because they presented healthy well-delineated borders. Aggregates of MO4 cells proved more suitable than suspensions or monolayer fragments because they were amenable to quantitation, were not traumatized or treated with enzymes during harvest. They allowed precise control of the initial site of contact with the heart tissue. The nature of the culture medium predominantly affected the growth of the MO4 population. This factor influenced the pattern of invasion quantitatively, but did not alter the invasive capacity of the MO4 cells itself. In cultures on adhesive substrates the interaction between the MO4 cells and the heart tissue was complicated by the fact that both also interacted with the aritficial substrate. Shaker cultures appeared better than comparable static cultures because they allowed better aeration and so delayed central necrosis.

An electron microscopic study of experimentally-induced HEV encephalitis.

A gnobiotic piglet, was inoculated intracerebrally with hemagglutinating encephalomyelitis virus (strain VW572). 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.

Influence of ethionine on the ultrastructural morphology of the porcine hepatocyte.

In the present study the morphological alterations induced by ethionine poisoning in the porcine liver were examined by electron microscopy. It was found that the most prominent ultrastructural changes were: accumulation of fat drops in the cytoplasm, disappearance of polysomes, fragmentation and vesiculisation of the endoplasmic reticulum, altered glycogen distribution and mitochondrial swelling. These closely resembled the pathological changes found in rat parenchymal cells under the same circumstances. In addition, moreover fat deposits were found in the nucleoplasm. The microbodies showed an altered morphology.

Electron microscopy of the intracellular development of bacteriophage MS2 in Escherichia coli.

The developmental cycle of bacteriophage MS2 in Escherichia coli was studied by means of ultramicrotomy and transmission electron microscopy. Application of a special fixation method made it possible to discern the individual phage particles from cellular ribosomes in the early infection stages and to follow the further development of the phage in the host bacterium. By means of the same techniques we have tried to obtain a better insight in the process of lysis of the infected cells.