Enhancement of Interleukin-4-Mediated Tumor Regression in Athymic Mice by In Situ Retroviral Gene Transfer

Abstract

Intratumoral grafting of genetically engineered cells that produce interleukin-4 (IL-4) has been shown to produce tumor regression as well as prolong survival of mice harboring intracerebral gliomas. We sought to determine whether retroviral-mediated gene delivery into tumor cells in situ resulted in enhanced tumor regression by IL-4. Two mouse fibroblast lines were obtained: they both secreted similar levels of IL-4 but one produced a retrovirus vector bearing the IL-4 gene (CRE-MFG-IL-4 cells), whereas the other did not (NIH3T3-IL-4 cells). In mixed transplantation assays in the subcutaneous flanks of athymic mice, CRE-MFG, IL-4 cells were more effective than NIH3T3-IL-4 cells in inhibiting the growth of rat C6 glioma cells (p < 0.005, ANOVA). Subcutaneous tumors injected with fibroblasts that produced a control retrovirus vector without producing IL-4 (CRE-MFG-LacZ cells) did not inhibit subcutaneous tumor growth. An intracranial assay was used to evaluate survival of athymic mice harboring intracranial gliomas. Three days after implanting rat C6 glioma cells into the right frontal lobes of athymic mice, NIH3T3-IL-4 cells (n = 10) or CRE-MFG-IL-4 cells (n = 10) were stereotactically inoculated into the tumor bed. The average survival of mice treated with CRE-MFG-IL-4 cells was 38 days (±2.4, SE), whereas that of mice treated with NIH3T3-IL-4 cells was 31 days (±0.8, SE) (p < 0.005, ANOVA; p < 0.001, log-rank analysis). The mean survival of control mice treated with CRE-MFG-LacZ cells was only 25 days (±0.7, SE) (p < 0.0001, compared to CRE-MFG-IL-4-treated mice). Histologic analysis of animals' tumors revealed extensive eosinophilic infiltrates in mice treated with the IL-4-producing cells (both NIH3T3-IL-4 and CRE-MFG-IL-4) but not in mice treated with IL-4 nonproducing cells (CRE-MFG-LacZ). This infiltrate started to decrease 3 weeks after fibroblast implantation, implying that its maintenance was needed for prolonged tumor regression. This study demonstrates that in situ retroviral-mediated transfer of the IL-4 gene enhances the anticancer effect of cells that produce the cytokine. Interleukin-4 (IL-4) has been shown to produce the regression of experimental brain tumors. This has been achieved by grafting cells genetically engineered to produce this cytokine. We have used a mouse fibroblast cell line that produces not only IL-4 but also a retrovirus vector that bears an IL4 transgene. As a control, we have made a mouse fibroblast cell line that produces the same levels of IL4 but that does not make retrovirus. We show that the IL4-retrovirus producer line is superior to the IL4-producing cells in achieving regression of subcutaneous brain tumors. In an intracerebral glioma model, the former cells produced more significant extensions in animal survival than the latter. These results indicated that the in situ retroviral gene delivery of IL-4 into brain tumors significantly extended the survival of mice compared to the in situ production of IL-4.