Rapid, in vivo, evaluation of antiangiogenic and antineoplastic gene products by nonviral transfection of tumor cells

Abstract

Using a nonviral, electroporation-based gene transfection approach, we demonstrate the efficient and consistent transfection of two poorly immunogenic tumor cell lines: B16F10 melanoma and renal carcinoma (RENCA). Three genes, IL-12, angiostatin (AS), and an endostatin:angiostatin fusion protein (ES:AS) were subcloned into a DNA plasmid containing EBNA1-OriP, which was then transfected into B16F10 and RENCA cells. Significant levels of protein were secreted into the culture supernatants of transfected cells in vitro. Transfected tumor cells were injected subcutaneously into mice. All the three transgenes were capable of significantly delaying and reducing the formation of primary B16F10 and RENCA tumors, as well as B16F10 lung metastases. By day 11 postinjection, all control mice that received either mock-transfected or empty vector DNA-transfected B16F10 tumor cells had developed large primary tumors. In contrast, mice that received IL-12-transfected B16F10 cells did not develop appreciable tumors until day 17, and these were significantly smaller than controls. Similar results were observed for the RENCA model, in which only one of the IL-12 mice had developed tumors out to day 31. Expression of AS or ES:AS also significantly delayed and reduced primary tumors. Overall, ES:AS was more effective than AS alone. Furthermore, 25% of the AS mice and 33% of the ES:AS mice remained tumor-free at day 17, by which point all control mice had significant tumors. Mouse survival rates also correlated with the extent of tumor burden. Importantly, no lung metastases were detected in the lungs of mice that had received either AS or ES:AS-transfected B16F10 tumor cells and significantly fewer metastases were found in the IL-12 group. The consistency of our transfection results highlight the feasibility of directly electroporating tumor cells as a means to screen, identify, and validate in vivo potentially novel antiangiogenic and/or antineoplastic genes.

Bacteriolytic therapy can generate a potent immune response against experimental tumors

“…To determine whether these results could be reproduced in another murine system, we repeated the experiment using RENCA cells, derived from a murine renal cell carcinoma that is also minimally to moderately immunogenic (16). Results obtained with this model were very similar to those observed with CT26.…”

Microbubble‐enhanced ultrasound exposure promotes uptake of methotrexate into synovial cells and enhanced antiinflammatory effects in the knees of rabbits with antigen‐induced arthritis

“…It does not require viral vector construction or virus preparation. Many reports describe electroporation as a technique for promoting electrochemotherapy through the uptake of MTX into cancer cells (17)(18)(19). We also showed that electrochemotherapy was effective in digital chondrosarcoma (20).…”

Angiogenesis meets immunology: Cytokine gene therapy of cancer

“…Weiss and co-workers used a non-viral, electroporation-based gene transfection approach and obtained efficient and consistent IL-12 transfection of two poorly immunogenic B16-F10 melanoma and renal carcinoma cell lines. Transfected tumor cells were then injected subcutaneously into mice and IL-12 was capable of significantly delaying and reducing the formation of tumors (Weiss et al, 2004).…”