Promoter-controlled infectivity-enhanced conditionally replicative adenoviral vectors for the treatment of gastric cancer.

BACKGROUND: Gastric cancer is the fourth most common malignancy worldwide. Adenoviral vectors (Ads) have been applied for gene therapy of various cancers because of their high transduction efficiency. However, the infectivity of gastrointestinal cancer cells is poor due to the limited expression of the Coxsackie-adenovirus receptor (CAR). In addition, few tumor-specific promoters (TSPs) have been characterized for this type of cancer. To overcome these problems, we proposed TSP-driven conditionally replicating adenoviruses (CRAds) with fiber modification for virotherapy of gastric cancer. METHODS: We assessed the expression profile of eight TSPs in gastric cancer cell lines and evaluated promising candidates in the context of CRAd cytocidal effect. Next, infectivity enhancement by fiber modifications was analyzed in the gastric cancer cell lines. Finally, we combined the TSP-driven CRAds of choice with the fiber modifications to augment the killing effect. RESULTS: Out of the eight TSPs, the midkine (MK) and cyclooxygenase-2 (Cox-2M and Cox-2L) promoters showed high transcriptional activity in gastric cancer cells. When these promoters were used in a CRAd context, Cox-2 CRAds elicited the strongest cytocidal effect. The greatest infectivity enhancement was observed with adenoviral vectors displaying 5/3 chimeric fibers. Likewise, Cox-2 CRAds with 5/3 chimeric fibers showed the strongest cytocidal effect in gastric cancer cell lines. Therefore, Cox-2 CRAds with 5/3 chimeric fiber modification showed good selectivity and infectivity in gastric cancer cells to yield enhanced oncolysis. CONCLUSIONS: Cox-2 CRAds with 5/3 chimeric fiber modification are promising for virotherapy of gastric cancer.

Blood-based screening and light based imaging for the early detection and monitoring of ovarian cancer xenografts.

We report a novel technology for in vivo early detection, identification, and monitoring of ovarian cancer in live mice leading to better treatment outcome. A genetic dualistic reporter system that uses an adenoviral (Ad) vector to transfer the genetic reporters to the ovarian cancer is described. Infection of the cancer cells leads to expression of one reporter that is detected in blood, namely, secreted human placental alkaline phosphatase (SEAP). A second reporter, namely, enhanced green fluorescent protein (GFP) is also delivered by the Ad, leading to expression at the site of ovarian cancer. The SEAP gene under control of the cytomegalovirus (CMV) promoter element is linked to the GFP gene with an IRES element. A diagnostic adenoviral vector (Ad) encoding the SEAP and GFP (Ad5-SEAP-GFP) is produced. Efficacy of newly developed diagnostic vector is tested in cell culture and animal models. SKOV3ip.1 cells are infected with Ad5-SEAP-GFP. Over time the cells are monitored for fluorescence and SEAP is also measured in the growth media supernatant. For animal experiments, SKOV3ip.1 cells are implanted first in nude mice either subcutaneously (SC) or intraperitoneally (IP) separately. After 4-7 days, the Ad5-SEAP-GFP is administered. Control mice do not receive any Ad vector. All mice are imaged with a fluorescent stereomicroscope after 24 h, and blood is collected for SEAP analyses. Increasing green fluorescence is detected in all SKOV3ip.1 cells infected with Ad5-SEAP-GFP, while SEAP levels in growth media increase over monitoring period. Expression of GFP in both SC and IP tumors is detected by 24 h in the live mice. At this time, the SEAP blood levels are more than 2-3 fold greater than blood levels of control group. GFP fluorescence and SEAP levels continue to increase in all mice that are injected with Ad5-SEAP-GFP until termination. Control mice (both SC and IP) do not express GFP or SEAP throughout the experiment. GFP contrast is necessary to differentiate between micro-sized early stage non-palpable ovarian tumor nodules and surrounding normal tissue. While the studies are conducted in mice, it is envisioned that the dual-based approach will eventually be translated into human applications for routine diagnosis and monitoring of ovarian cancer when an ovarian cancer specific promoter will be available. Due to the thickness of the abdominal wall in human laparoscopy or laparotomy will be necessary. This system will provide gynecologic oncologists with a more effective tool for treating patients. The blood-based screening assay provides a quick test to determine the presence of the ovarian cancer at its earliest stage. The location of the ovarian cancer is afforded by the light-based imaging component, which represents a new and improving technology with tremendous advantages of sensitivity and spatial resolution to localize micro-sized tumor nodules. The novelty of the dualistic system is the linkage of blood-based reporter screening as a selection criteria for subsequent light-based imaging procedures. This combination will lead to an accurate and widely applicable method for the early detection and monitoring of ovarian cancer, especially in high-risk women

Gamma camera dual imaging with a somatostatin receptor and thymidine kinase after gene transfer with a bicistronic adenovirus in mice.

PURPOSE: To compare two systems for assessing gene transfer to cancer cells and xenograft tumors with noninvasive gamma camera imaging. MATERIALS AND METHODS: A replication-incompetent adenovirus encoding the human type 2 somatostatin receptor (hSSTr2) and the herpes simplex virus thymidine kinase (TK) enzyme (Ad-hSSTr2-TK) was constructed. A-427 human lung cancer cells were infected in vitro and mixed with uninfected cells at different ratios. A-427 tumors in nude mice (n = 23) were injected with 1 x 10(6) to 5 x 10(8) plaque-forming units (pfu) of Ad-hSSTr2-TK. The expressed hSSTr2 and TK proteins were imaged owing to internally bound, or trapped, technetium 99m ((99m)Tc)-labeled hSSTr2-binding peptide (P2045) and radioiodinated 2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl-5-iodouracil (FIAU), respectively. Iodine 125 ((125)I)-labeled FIAU was used in vitro and iodine 131 ((131)I)-labeled FIAU, in vivo. The (99m)Tc-labeled P2045 and (125)I- or (131)I-labeled FIAU were imaged simultaneously with different window settings with an Anger gamma camera. Treatment effects were tested with analysis of variance. RESULTS: Infected cells in culture trapped (125)I-labeled FIAU and (99m)Tc-labeled P2045; uptake correlated with the percentage of Ad-hSSTr2-TK-positive cells. For 100% of infected cells, 24% +/- 0.4 (mean +/- SD) of the added (99m)Tc-labeled P2045 was trapped, which is significantly lower (P <.05) than the 40% +/- 2 of (125)I-labeled FIAU that was trapped. For the highest Ad-hSSTr2-TK tumor dose (5 x 10(8) pfu), the uptake of (99m)Tc-labeled P2045 was 11.1% +/- 2.9 of injected dose per gram of tumor (thereafter, dose per gram), significantly higher (P <.05) than the uptake of (131)I-labeled FIAU at 1.6% +/- 0.4 dose per gram. (99m)Tc-labeled P2045 imaging consistently depicted hSSTr2 gene transfer in tumors at all adenovirus doses. Tumor uptake of (99m)Tc-labeled P2045 positively correlated with Ad-hSSTr2-TK dose; (131)I-labeled FIAU tumor uptake did not correlate with vector dose. CONCLUSION: The hSSTr2 and TK proteins were simultaneously imaged following dual gene transfer with an adenovirus vector.