Abstract
An increasing number of reports highlight the fact that tumour cells are able to give rise in vitro to immunogenic variants, which are defined in vivo as being non tumorigenic, tum-. We have observed the emergence of immunogenic variants, derived from a primary nickel-induced rat rhabdomyosarcoma established in culture (RMS 9-4/0), resistant to treatment with the chloronitrosourea, chlorozotocin (CZT) (R-lines). They were separated from the whole population of cells by a cloning procedure. Furthermore, we demonstrate that the cloning procedure by itself allows the isolation of tum- variant designated as C-lines. In both cases, the tum- phenotype was observed after s.c. injection of cells into syngeneic rats with a broad range of R9 or C8 cells (10(4) to 10(7). This characteristic was inherited in a stable manner. Athymic mice developed tumours of rat rhabdomyosarcoma origin when grafted with 10(5) cells. Immunization of rats with one R variant (R9) tum- protected the rats grafted with the parental RMS 9-4/0 cells against metastatic invasion of the lungs, but not against local tumour growth, and rats grafted with a CZT-resistant tum+ cell variant S4T (in vivo-derived) against its hepatic and pulmonary metastases, while the local tumour progressed as usual. Immunization of rats with one C variant (C8) tum- cells did not protect them against either metastases or local growth of the implanted tumours. Both R and C lines cells became progressively resistant to NK- and macrophage-induced cytotoxicity. Splenic lymphocyte transfer from immune rats into nude mice, i.e., the Winn test, showed a complete degree of protection against C8 or R9 tumour growth. We conclude that two different antigenicities were revealed, one common to R9 and C8 cells in relation with their selection procedure by repeated cloning. Another antigenicity appeared in the R9 line, selected by CZT-resistance. The anti R9 cell immunization against CZT-resistant tum+ S4T could argue in favour of CZT action in the acquisition of R9 cell antigenicity. More likely, an amplification of antigens rather than induction of a new antigen could explain the protection of anti R9 immunized rats against parental tumour metastases.
Highlights
Summary An increasing number of reports highlight the fact that tumour cells are able to give rise in vitro to immunogenic variants, which are defined in vivo as being non tumorigenic, tum
We have observed the emergence of immunogenic variants, derived from a primary nickel-induced rat rhabdomyosarcoma established in culture (RMS 9-4/0), resistant to treatment with the chloronitrosourea, chlorozotocin (CZT) (Rlines)
We demonstrate that the cloning procedure by itself allows the isolation of tum- variant designated as C-lines
Summary
AnimalsInbred Wistar AG female rats 8-12 weeks old were obtained fromfrotmh~~e~~~s~pp~eccitffhiccppatthoogeen--frreeebbreeeddning faaciliityyoof thheInstitut de Recherches Scientifiques sur le Cancer.Nude rats with a Wistar AG genetic background were obtaired backcross.andthTrey from MRC(UK)atthe10th wobetraeinberdedfrtoomtththehee12MthRbCack((UcKro)ssatantdheth1e0tphrobgaecnkcyyrowsesr.eThey u used in thesexeperiments. Inbred Wistar AG female rats 8-12 weeks old were obtained fromfrotmh~~e~~~s~pp~eccitffhiccppatthoogeen--frreeebbreeeddning faaciliityyoof thhe. Institut de Recherches Scientifiques sur le Cancer. Chlorozotocin (2-(3-(2-chloroethyl)-3 nitrosoureido)-D-glucopyranose); CAS: 54749-90-5), a nitrosourea and alkylating agent, was provided by the Division of Cancer Treatment, National Cancer Institute, Bethesda, MD. Cell lines and establishment of variant cell lines. RMS 9-4/0 parental cells were derived from a primary nickel-induced tumour in a Wistar AG inbred rat, as previously described (Sweeney et al, 1982). Cells and resistant variants were maintained in Dulbecco's modified Eagle's medium (DMEM, H21, Grand Island Biological Co. Glasgow, UK) supplemented with 10% heat-inactivated foetal calf serum (FCS) and subcultured twice weekly by trypsinization. Cells were always free of mycoplasma contamination, as assessed by Hoechst staining (Chen, 1977)
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