Abstract
Gene-transfected tumour cells were used to cure mice bearing lung metastases by the parental, non-transduced mammary adenocarcinoma (TSA-pc). Repeated subcutaneous (s.c.) administrations of mitomycin C (MitC)-treated interferon gamma (IFN-gamma) transfectants induced a 90% inhibition in the number of lung metastases. Therapeutic effect required an intact T-cell response, as shown by the lack of efficacy in nude mice. Autocrine stimulation by IFN-gamma induces specific modifications in the phenotype of transfectants that acquire a high metastatic ability and show a high expression of IFN-responsive genes; these two features were exploited to design two experimental protocols to obtain an improvement of the therapeutic effect. The increased metastatic ability of IFN-gamma transfectants was used to deliver IFN-gamma selectively to the lungs of mice bearing TSA-pc pulmonary metastases. A significant therapeutic effect was obtained when TSA-pc experimental metastases were treated by repeated intravenous (i.v.) injections of MitC IFN-gamma transfectants. Since i.v. administrations of IFN-gamma transfectants did not induce immune memory, the therapeutical effect appeared to depend on the inflammatory-like response activated by local IFN release. To exploit the autocrine stimulation of IFN-sensitive genes an IFN-gamma transfectant clone was subjected to a second transfection with an allogeneic class I MHC gene (H-2K(b) or H-2D(h)). IFN-gamma plus MHC double transfectants maintained IFN-gamma release, showed a very high expression of the MHC gene products, stimulated both macrophages and T cells, and were less tumorigenic in immunocompetent mice than the parent IFN-gamma clone. Therapeutic efficacy of double transfectant IFN-gamma plus H-2D(b) cells against TSA-pc was superior to single transfectants, showing that the reaction elicited by genetically engineered cells can be selectively tuned to increase therapeutic efficacy.
Highlights
Cells were cultured in Dulbecco's modified eagle medium (DMEM; Gibco, Paisley, UK), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco); cultures were maintained at 37°C in a humidified atmosphere of 5% carbon dioxide in air
TSA-pc is a highly malignant tumour, and expression of the IFN-j; gene did not abolish completely the tumorigenicity of clones TSA-IFN;500 and TSA-IFN-.6""., mitomycin-C-treated transfectants treated with 60 jig ml-' mitomycin-C (Sigma, (MitC) cells were used for therapeutic administrations
It should be noted that allogeneic major histocompatibility complex (MHC) expression of double transfectants was 70 times higher than expression of cells transfected with the MHC gene alone in the absence of autocrine induction by interferon y (IFN-y)
Summary
TSA-pc is a tumour cell line we derived from a spontaneous mammary adenocarcinoma of the BALB/c strain; TSA-pc cells give rise to moderately differentiated, non-capsulated invasive tumours, which are highly metastatic and poorly immunogenic in syngeneic mice (Lollini et al, 1993). (which release 500 and Either before or after the TSA-pc challenge, mice received 6000 IU ml-' IFN-; respectively), mitomycin-C-treated transfectants treated with 60 jig ml-' mitomycin-C (Sigma, (MitC) cells were used for therapeutic administrations. Italy) at 37°C for 45 min to abolish their residual cells retain for several days the ability to release IFN-j;, as tumorigenicity (Allione et al, 1994). Therapeutic vaccinations started 1 day after TSA-pc potential of IFN-; clones was mainly caused by the local challenge and consisted of six injections of 1 x 106 MitC activity of macrophages. MitC TSA-pc cells and tested for cytotoxic activity. 5"Crlabelled TSA-pc cells were used to evaluate 4 h release in the presence of effectors obtained as above, but very low values of cytotoxicity were obtained owing to the resistance to lysis of
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