Previous studies demonstrated that growth in DBA/2 mice of MDW4, a wheat germ agglutinin-resistant (WGA9 mutant of the highly metastatic MDAY-D2 DBA/2 mouse tumor, led to the emergence of WGA-sensitive (WGAS) revertants having higher ploidy levels at the site of inoculation as well as at distant visceral metastases. The results implied that MDW4 was nonmetastatic but progressed to become metastatic in vivo only after a cellular change took place which was accompanied by extinction of the WGAr phenotype and acquisition of a higher number of chromosomes. Results presented here provide strong and direct evidence for the underlying mechanism being spontaneous cell fusion in vivo between the MDW4

The evolution towards more aggressive and autonomous behaviour of many cancerous tumours, often referred to as tumour progression, is thought to stem from the development of heterogeneity within the tumour cell population, combined with the continuous selection of progressively more malignant cellular phenotypes. During the course of the disease, the tumour cells show multiple phenotypic changes in a stepwise, but apparently random fashion, becoming more anaplastic, increasingly independent of growth controls and more metastatic. Several laboratories, including our own, have analysed aspects of tumour heterogeneity and cancer metastasis by selecting and studying the properties of lectin-resistant (LecR) membrane mutant tumour sublines; in a few cases, such variants have been claimed to be less tumorigenic or metastatic than the parental cells from which they were derived. We have attempted to study the factors involved in the reestablishment of tumour heterogeneity by monitoring the stability in vivo of LecR phenotypes of metastatic tumour cells after injection of cloned LecR tumour cells. We now report that spontaneous metastases arising after a subcutaneous (s.c.) injection of cells from variant tumour lines selected from a highly metastatic DBA/2 mouse tumour known as MDAY-D2, and which are stably resistant in tissue culture to wheat germ agglutinin (WGA), no longer carry the WGA-resistant (WGAR) phenotype. The results demonstrate that WGAR tumour cells do not metastasize, but rather, 'revertants' for the WGAR phenotype, which presumably were generated in vivo after injection, were the cells actually capable of metastatic growth.

Treatment of normally tumorigenic murine tumor cell lines in vitro with chemical mutagens followed by cloning of the surviving cells, results in the selection, at extraordinarily high frequencies (anywhere from <1% to >90%), of clones unable to grow progressively in normal syngeneic mice (1-7). Such clones, which are phenotypically stable in culture over a period of several weeks or months, have been designated by Boon and his colleagues (1-6) as "tum-" (nontumorigenic in normal hosts). ~ They have been derived using such standard mutagens as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and ethyl methanesulfonate (EMS), from at least eight different murine tumors of viral, chemical, or spontaneous origin (1-7). Because the turn-clones will grow readily in highly immunosuppressed recipients, e.g., X-irradiated or nude mice (1-8), the turn-phenotype appears to have an underlying immunological basis, a view confirmed by detailed in vitro studies of T cell-mediated cytotoxicity (CMC).These studies have also shown that each turn-clone derived from a particular mutagenized parent line possesses an individual tumor-specific antigen distinct from the new antigen found on any other turn-clone, i.e., there is a startling degree of tumor antigen polymorphism (1-7). Of considerable interest is that this holds true even for totally non-immunogenic tumors of spontaneous origin (5-7) and as such, has very broad and important implications for the field of tumor immunology, especially in relation to the controversies surrounding the antigenic and immunologic status of neoplastic cells (9-11). Nevertheless, very little is known about how mutagens can cause such drastic and heritable changes in the behavioral properties of tumors in vivo, and at such high frequencies.

No abstract

Single-step mutants were isolated from the murine metastatic MDAY-D2 cell line after selection in toxic concentrations of wheat-germ agglutinin. They were partially characterized by measuring their relative level of resistance to WGA, PHA, Con A, RIC, and LCA (Lec phenotype), and by comparing their karyotype and their ability to produce metastases upon transplantation into syngeneic DBA/2 mice. Based on their Lec phenotype, a total of 19 independent isolates were ranked into 10 distinct classes. Among them, two EMS-induced mutants were nontumorigenic (Lec II, Lec III), one nonmetastatic (Lec IV), and one spontaneous mutant (Lec I) failed to produce blood-borne metastases. Other spontaneous mutants belonging to Lec I, Lec II, and other classes were as metastatic as their parents. The Lec IV phenotype was found to segregate independently from metastatic potential in somatic hybrids. Metastatic ability was recovered in mutants expressing the Lec IV phenotype, after further selection for resistance to RIC. Our results strongly suggest that the loss or reduction of the invasive property of tumor cells is associated with only few Lecr1 phenotypes and, therefore, that a restricted number of cell surface glyconjugates are essential for this particular function.