Interaction of B-cell hybridomas with fibroblast or hepatocyte monolayers in vitro and their metastatic behaviour in vivo.

Using an in vitro monolayer assay (MIA) we analyzed the invasive behaviour of a panel of B-cell hybridomas prepared by the fusion of non-invasive, non-metastatic NSO plasmacytoma cells and normal murine B-cells. Interaction of these hybridomas with fibroblast-like monolayers consisted mostly of adhesion on top of the monolayers, whereas only a fraction of these cells penetrated through the monolayer. This is in sharp contrast with the highly invasive properties displayed by T-cell hybridomas. Whereas T-cell hybridomas highly infiltrated monolayers of rat hepatocyte in vitro, B-cell hybridomas neither adhered to nor infiltrated hepatocyte monolayers. We found a good correlation between the degree of adhesion of B-cell hybridomas to fibroblast-like monolayers and their metastatic capabilities upon i.v. injection into syngeneic animals. Unlike T-cell hybridomas which formed diffuse metastasis in liver and spleen, B-cell hybridomas generated nodular metastatic lesions. . When normal LPS-stimulated B-lymphocytes were tested in the fibroblast-MIA, only part of the population infiltrated the monolayers. This again contrasts with T-lymphocytes where a majority of the cells penetrated through the monolayers. These results suggest that (i) B-lymphocytes express invasive properties, albeit to a lesser extent than T-lymphocytes, (ii) non-invasive B-lymphoma cells can acquire invasiveness following cell fusion with a normal B-cell, (iii) these invasive properties contribute to the malignancy of the hybridomas when tested in recipient animals.

Experimental analysis of the metastatic phenotype of malignant leukocytes.

The metastatic phenotype (M+) is determined by the expression of the invasive (I+) and growth (G+) phenotypes at various sites along the metastatic pathway. The G, I and M phenotypes are not confined to tumor cells and may be expressed by normal cells, such as leukocytes, under certain physiological conditions. The study of the I and M phenotypes of leukocytes may yield valuable information on common mechanisms, underlying invasion and metastasis of normal and malignant cells. In this review we summarize experimental approaches that were developed for the analysis of the I and M phenotypes of normal and malignant leukocytes.

Identification of T-cell antigens expressed by metastatic T-cell hybridomas and lymphomas.

Using the murine BW5147 tumor model system, we have identified 3 MAbs that discriminate between metastatic and non-metastatic BW5147-derived T-cell hybridomas and BW5147-unrelated T-lymphomas. The 3 rat MAbs appear to recognize an identical membrane-associated sialoglycoprotein with an approximate molecular weight of 95-100 kDa. We thus defined "metastatic T-cell hybridoma" antigens (MTH-Ags) that are also expressed on normal T-lymphocytes. No correlation was found between the expression of the MTH Ags and in vitro invasive behavior of normal and malignant cells. Neither did we find any relationship between organ specificity of i.v. inoculated tumor cells and their MTH-Ags expression. It thus remains unclear whether our MTH-Ags are functionally involved in the metastatic process, or whether their expression is only incidentally related to the metastatic potential.

Suggestive evidence that genes controlling invasion and metastasis of T-cell lymphomas are located on mouse chromosome 3.

Cell lines differing in their malignant potential have been derived from the murine BW5147 T-cell lymphosarcoma. To evaluate the involvement of chromosomal aberrations in tumor progression within this model, we have analyzed the karyotypes and the in vitro invasiveness of 13 related nonmetastatic and metastatic variants. Giemsa banding revealed the presence of several marker chromosomes, one of which was of particular importance. Depending on the cell line, four variants of this marker I were found: Marker Ia corresponds to two translocated chromosomes 3, marker Ib is a deleted Ia marker, marker Ic is a Ib translocated to small unidentified chromosome fragment, and marker Id is a further deleted Ib marker. The Ia and Id markers were characteristic for the noninvasive, nonmetastatic lines, whereas the Ib and Ic markers predominated in the invasive, metastatic variants. The results suggest that metastasis-enhancing genes are located between the D and FI band of mouse chromosome 3 and that metastasis-suppressing genes are located between the FI and H band of the same chromosome.

Dual effects of pertussis toxin on in vitro invasive behavior of metastatic lymphoma variants.

A simple monolayer invasion assay (MIA) was recently developed using confluent fibroblastic cells as a target and variants of the BW5147 murine T-cell lymphoma as invading cells. Metastatic variants were consistently invasive in the MIA whereas non-metastatic cells were not. In this paper it is reported that pertussis toxin (PT) treatment of a highly metastatic and invasive variant caused a marked delay of invasion in the MIA at concentrations from 50 pg/ml upwards. Surprisingly, PT treatment of the non-metastatic, non-invasive parental BW5147 cells induced a moderate but significant level of invasion. Morphometric analysis showed that PT provoked an increased pseudopodal activity in cells in which it also caused increased invasive potential, and a decreased motility in cells with decreased invasiveness. This finding strengthens the perception that invasive potential and the capability to perform shape changes are related characteristics in these lymphoma cells.

Syngeneic in vivo passage of the murine BW 5147 lymphoma results in the expression of a stable metastatic phenotype.

BW 5147 lymphoma cells are non-invasive tumor cells which do not generate experimental metastases following i.v. inoculation. In contrast, s.c. and intra-splenic (i.s.) growth of BW cells resulted in widespread colonization of liver and spleen. Cells derived from either s.c. tumors or metastatic lesions did generate metastases after i.v. administration. The capacity of these tumor-derived BW cells to disseminate via blood-borne cells was irreversible and stable, indicating that one in vivo passage of BW cells results in the generation of new, metastatic BW variants. Concomitantly, these variants exhibited an inherent invasive potential as manifested by their capacity to infiltrate in vitro monolayers of hepatocytes and fibroblasts. The BW variants expressed new membrane markers such as H-2 antigens, the Lyt 1.2 T-cell differentiation antigen and the MTH antigen (a newly defined membrane antigen expressed predominantly on murine metastatic T-cell lymphomas and mature T lymphocytes). This phenomenon was observed with both cloned and uncloned BW populations, suggesting that an inductive rather than a selective mechanism accounts for the transition of BW cells towards a more malignant phenotype. These observations confirm the concept that local factors at the growth site of a tumor might influence the metastatic behavior of that tumor, possibly via induction of silent differentiation programs.