Metastatic properties of distinct phenotypic classes of lectin-resistant mutants isolated from murine MDAY-D2 cell line.

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.

Characterization of in vitro immunoselected variants from a highly metastatic murine tumor for alterations in malignant behavior in vivo.

A new Ly-6.2- antigen-loss variant (called L61 -M1) of the highly metastatic DBA/2 mouse (Ly-6.2+) MDAY-D2 tumor has been obtained by means of a monoclonal anti-Ly-6.2 antibody in an in vitro immunoselection technique. Whereas L61 -M1 grew poorly when inoculated subcutaneously into the syngeneic host, it grew and metastasized in a similar way to the parental MDAY-D2 tumor when inoculated into immunosuppressed, athymic nude mice. L61 -M1 as well as another Ly-6.2- variant of the same MDAY-D2 tumor (called L61 ) which is poorly metastatic in the syngeneic host salvaged exogenous fucose into glycoproteins and glycolipids at rates 5.5 and 7.8 times that of the parental MDAY-D2 line. In contrast, the Ly-6.2- variants exhibited a 50-70% decrease in the incorporation of exogenous mannose into glycoproteins and glycolipids. L61 -M1 and L61 also exhibited alterations in the structures of the oligosaccharide moieties linked to the cell surface glycoproteins and/or glycolipids. Thus, the in vitro immunoselection technique can be used to obtain a panel of variants with stable phenotypic alterations in their growth and metastatic capacities. Such mutants may, like previously described lectin-resistant mutants, be useful in studying the contribution of cell surface glycoproteins and glycolipids to tumorigenicity and metastasis.

Selection of strongly immunogenic "tum-" variants from tumors at high frequency using 5-azacytidine.

Highly immunogenic "tum-" (non-tumorigenic in normal syngeneic hosts) clonal variants can be selected from a variety of poorly immunogenic and highly tumorigenic mouse cell lines at very high frequencies (e.g., greater than 80%) after treatment in vitro with chemical mutagens such as ethyl methanesulfonate (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We herein demonstrate that the same result can be obtained with the poorly mutagenic cytidine analogue, 5-azacytidine, a strong DNA hypomethylating agent. 5-Azacytidine and EMS were equally and comparably effective, or ineffective, in inducing tum- variants from three different highly tumorigenic mouse cell lines. Like mutagen-induced tum- variants, those obtained after 5-azacytidine treatment generated usually strong cytolytic T lymphocyte (CTL) responses in vitro, and could grow in immunosuppressed (nude mouse) hosts. However, pretreatment of the tumor cell lines with 5-azacytidine did not cause significant increases in mutations at several independent drug-resistant gene loci, whereas EMS did. It is known that treatment of cells with 5-azacytidine can induce transcriptional activation of "silent" genes through a reduction of DNA 5-methylcytosine content, a process that can also be effected by mutagenic DNA alkylating agents such as EMS and MNNG. We therefore hypothesize that an "epigenetic" mechanism (DNA hypomethylation) leading to activation and expression of genes coding for potential tumor antigens is involved in the generation at high frequency of tum- variants after "mutagen" treatment. The implications of these findings to mechanisms of tumor progression and the generation of tumor heterogeneity are discussed.

Genotypic and phenotypic evolution of a murine tumor during its progression in vivo toward metastasis.

To follow the cellular progeny of the multiple-drug-marked benign murine tumor cell line MDW4 during its progression in vivo toward metastatic spread in DBA/2 mice, the following parameters were analyzed: retention of the drug-resistant markers ouabain resistance (OuaR) and thioguanine resistance (ThgR), lectin-resistance pattern (WGAR), and the karyotype of cell populations (and clones derived from these cells) removed at intervals from the solid tumor growing at the site of inoculation, as well as distant metastatic nodules. It was determined that the initially homogeneous inoculum composed of OuaR, ThgR, and WGAR hypotetraploid cells (mode: 68 +/- 2 chromosomes) was gradually overgrown and replaced by a new population of cells that were either OuaR or ouabain-sensitive but that became thioguanine-and lectin-sensitive and hyperploid (mode: 95 +/- 5). Regardless of the composition of the individual drug marker combinations, only cells with high chromosome contents were found to be able to disseminate to distant visceral organs and to rapidly produce metastases upon sc or iv reinjection. The presence of the same number of metacentric chromosomes in metastatic cells as in MDW4 and the coextinction of two recessive drug-resistant markers (WGAR and ThgR) suggested that cells endowed with invasive-metastatic potential represent the product of spontaneous somatic hybridization between the original nonmetastatic MDW4 cells and normal host cells of unknown origin. Such a fusion was followed by more or less extensive chromosome segregation that accounts for the karyotype mosaicism and the occasional drug marker heterogeneity identified in cell populations of metastatic nodules.

Spontaneous fusion in vivo between normal host and tumor cells: possible contribution to tumor progression and metastasis studied with a lectin-resistant mutant tumor.

Previous studies demonstrated that growth in DBA/2 mice of MDW4, a wheat germ agglutinin-resistant (WGAr) 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 (WGAr) tumor cells and normal host cells, at least some of which are of bone marrow origin. Thus, growth of the H-2d MDW4 tumor cells in (C3H X DBA/2)F1 (H-2k X H-2d) or (C57BL/6 X DBA/2)F1 (H-2b X H-2d) mice led to the appearance of WGAs revertants bearing the H-2k or H-2b major histocompatibility complex antigens associated with the C3H or C57BL/6 parental strains, respectively. Similarly, WGAs revertants of MDW4 were found to express H-2k antigens after growth in CBA/HT6T6 (H-2k) leads to DBA/2 bone marrow radiation chimeras. Attempts to mimic the in vivo hybridization process were successful in that in vitro somatic cell fusion between an ouabain-resistant (OuaR), 6-thioguanine-resistant (Thgr) derivative of the MDW4 mutant and either normal bone marrow or spleen cells resulted in loss of the WGAr phenotype in the hybrids (thus showing its recessive character) and increased malignant properties in vivo. An analysis of spontaneous frequencies of re-expression of various drug resistance genetic markers in several hybrid metastatic cells was also consistent with chromosome segregation of the sensitive alleles. The results show that tumor progression and the emergence of metastatic cell variants could arise as a consequence of tumor X host cell fusion followed by chromosome segregation. We also discuss the possibility that this type of event may normally be a very rare one during the growth of tumors, the frequency of which can be artificially amplified by the use of certain classes of lectin-resistant mutants carrying particular cell surface alterations.

An examination of tumor antigen loss in spontaneous metastases.

Metastases arising from a subcutaneous injection of the DBA/2 tumor, MDAY-D2, as well as four drug-resistant variants (either wheat germ agglutinin-resistant, ouabain-resistant, or both, i.e., WGAR/OuaR) of MDAY-D2, were examined for the presence of a tumor-associated antigen (TAA). Of 15 mice examined, tumor antigen-loss variants were detected in only 1 animal. These antigen-loss metastases arose in a mouse injected with the WGAR variant called MDW4. The tumor at the site of inoculation retained the TAA, whereas all four of the metastases removed from liver, spleen and other tissues were antigen-loss variants. The antigen-loss variants were not killed by cytotoxic T cells (CTL) directed against the TAA of the parental tumor, did not competitively inhibit CTL lysis of MDW4 targets in a 'cold target' inhibition test, and were not able to elicit a CTL response. In vivo immunization-protection (challenge) experiments also showed that the metastases did not express the TAA of MDAY-D2. Unlike the WGAR phenotypes, which were lost in all spontaneous metastases recovered from MDW4-injected mice, loss of the TAA appeared to be an uncommon event. Antigen-loss tumor cell variants are discussed in terms of their relevance to metastasis, and in regard to their use in the study of T cell-mediated cytotoxicity of tumor cell populations.

Membrane-associated alterations detected in poorly tumorigenic lectin-resistant variant sublines of a highly malignant and metastatic murine tumor.

A number of wheat germ agglutinin-resistant (WGAR) variants of a highly malignant and metastatic mouse tumor (called MDAY-D2) were selected. Two of these, MDW1 and MDW3, were poorly tumorigenic in the normal DBA/2 host but grew well in highly immunosuppressed recipients. In contrast, MDAY-D2, MDW4, and MDW5 were all highly tumorigenic in both normal and immunosuppressed hosts. Analysis of the WGAR variants by cytotoxic T-cell testing did not reveal any evidence for the acquisition of new tumor antigens by the MDW1 or MDW3 variant sublines, although they provoked a significantly stronger T killer cell response than did MDAY-D2, MDW4, or MDW5. Further studies indicated that the rate of tumor cell surface shedding in vitro correlated strongly with relative tumorigenicity and, furthermore, that changes in the cytoskeletal structure of MDW1 and MDW3 may have contributed to their reduced rate of shedding.