CWR22 Xenograft as an Ex Vivo Human Tumor Model for Prostate Cancer Gene Therapy

Abstract

Lack of well-defined relevant in vivo or in vitro tumor models is one of the major limitations in assessing candidate therapeutic regimens, especially gene therapy, for prostate cancer. Since gene therapy is emerging as a potentially powerful therapeutic modality, it is desirable to evaluate this approach for the treatment of human prostate cancer. We sought to establish a relevant ex vivo tumor model for gene therapy studies of human prostate cancer. We constructed and established a transgenic human tumor model consisting of three major components: 1) human primary prostate cancer cells, CWR22, reactivated for growth after storage in liquid nitrogen; 2) a collagen gel ex vivo tissue culture system useful for short-term maintenance and manipulation of CWR22 cells under in vitro experimental conditions; and 3) a high-velocity, particle-mediated gene transfer system that is highly efficient in the ex vivo transfection of target cells. Prostate-specific antigen (PSA) levels in the cell culture media were monitored after transfecting CWR22 cells with candidate therapeutic genes, including the cytokines human interleukin 2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), both as complementary DNAs [cDNAs]). CWR22 cells, transfected with firefly luciferase cDNA as a reporter gene, served as control cells for cytokine gene expression. CWR22 cells, transfected with the bacterial beta-galactosidase cDNA as a reporter gene, were used to assess the efficiency of gene transfer. Transcription of each of the cDNAs was driven by the cytomegalovirus (CMV) early gene promoter. The three-dimensional organization of tumor cells and functional characteristics of human prostate cancers were maintained in this ex vivo model of prostate cancer. Candidate therapeutic genes, CMV-IL-2 and CMV-GM-CSF, were expressed at peak levels of up to 38 ng of protein per 10(6) cells every 24 hours. IL-2 and GM-CSF secretion was sustained at approximately 40%-50% of peak levels during the entire experimental period (9-10 days in culture). At 7 days after gene delivery, a more than twofold reduction in the secretion of PSA was detected in the IL-2 (3.8 +/- 1.3 ng/10(4) cells every 24 hours [mean +/- standard deviation]) or GM-CSF (4.0 +/- 1.7 ng/10(4) cells every 24 hours) cDNA transfected cells as compared with the control cells transfected with luciferase cDNA (9.3 +/- 1.0 ng/10(4) cells every 24 hours). Up to 10% of the cells transfected with beta-galactosidase cDNA expressed measurable beta-galactosidase activity. This study demonstrated an efficient, rapid, and reliable system for gene transfer and expression in primary human prostatic carcinoma cells maintained in a collagen gel culture system. Our findings suggest a broad application of this CWR22 xenograft primary culture system as an ex vivo tumor model for the evaluation and characterization of various candidate therapeutic genes for human prostate cancer gene therapy, including a cytokine gene-modified tumor vaccine strategy.