Efficacy of CD46-targeting chimeric Ad5/35 adenoviral gene therapy for colorectal cancers

Abstract

CD46 is a complement inhibitor membrane cofactor which also acts as a receptor for various microbes, including species B adenoviruses (Ads). While most Ad gene therapy vectors are derived from species C and infect cells through coxsackie-adenovirus receptor (CAR), CAR expression is downregulated in many cancer cells, resulting inefficient Ad-based therapeutics. Despite a limited knowledge on the expression status of many cancer cells, an increasing number of cancer gene therapy studies include fiber-modified Ad vectors redirected to the more ubiquitously expressed CD46. Since our finding from tumor microarray indicate that CD46 was overexpressed in cancers of the prostate and colon, fiber chimeric Ad5/35 vectors that have infection tropism for CD46 were employed to demonstrate its efficacy in colorectal cancers (CRC). CD46-overexpressed cells showed a significantly higher response to Ad5/35-GFP and to Ad5/35-tk/GCV. While CRC cells express variable levels of CD46, CD46 expression was positively correlated with Ad5/35-mediated GFP fluorescence and accordingly its cell killing. Injection of Ad5/35-tk/GCV caused much greater tumor-suppression in mice bearing CD46-overexpressed cancer xenograft compared to mock group. Analysis of CRC samples revealed that patients with positive CD46 expression had a higher survival rate (p=0.031), carried tumors that were well-differentiated, but less invasive and metastatic, and with a low T stage (all p<0.05). Taken together, our study demonstrated that species B-based adenoviral gene therapy is a suitable approach for generally CD46-overexpressed CRC but would require careful consideration preceding CD46 analysis and categorizing CRC patients.

Keywords: CD46; adenovirus; colorectal cancer; gene therapy.

Figure 1. Gene transduction efficacy of Ad5/35 is enhanced in CD46-expressing cells

A. Western blot analysis of CD46 expression in parental rodent BHK cells or BHK-CAR and BHK-CD46 cells which ectopically express the Ad receptors CAR and CD46, respectively. B. Flow cytometric analysis of CD46 expression in BHK, BHK-CAR and BHK-CD46 cells. C. Transduction analysis of BHK, BHK-CAR and BHK-CD46 cells using different doses of Ad5/35-GFP. Cells were analyzed for GFP reporter expression 24 hrs post transduction by flow cytometry. Numbers in parentheses indicate percentage of GFP positive cells. D. Cell killing assay in BHK, BHK-CAR and BHK-CD46 cells transduced with Ad5/35-tk and treated with GCV for 4 days. Cytotoxicity was analyzed by the MTT assay. Error bars represent SEM. Statistics: C-D, p<0.01 by 2-way ANOVA.

Figure 2. CD46 expression analysis and Ad5/35-mediated gene transduction in colorectal cancer cell lines

A. CD46 expression in colon cancer cell lines was analyzed by Western blot analysis. B. CD46-positive cells were detected by flow cytometry in response to different dose of Ad5/35-GFP. C-E. Colon cancer cells were applied to flow cytometry to demonstrate intensity of CD46 expression (C) or Ad5/35-mediated reporter transduction (D). Percentage of cells double-stained with CD46 and GFP was shown in E. Error bars represent SEM. Statistics: B, p<0.01 by 1-way ANOVA and post-hoc Tukey test; C-D, p=0.267 by 2-way ANOVA.

Figure 3. Ad5/35-tk-mediated enhancement of in vitro cytotoxicity in CRC cells

Cells were seeded onto 24-well plates and incubated overnight. Subsequently, cells were transduced with Ad5/35-tk at the indicated MOIs ranging from 0 to 20. Varying concentrations of GCV were added the next day and MTT in vitro proliferation assays were performed 5 days post infection. DLD-1, Caco-2, and HCT-116 cells more effectively responded to the cytotoxic effect of Ad5/35/GCV than HT-29 and SW620 cells. Error bars represent SEM. Statistics: p<0.01 by 2-way ANOVA.

Figure 4. CD46 promotes Ad5/35-tk-mediated cytotoxicity for tumor growth in vivo

A-B. Cells were transduced with Ad5/35-tk followed by GCV treatment. MTT in vitro proliferation assays were performed 5 days post infection. C-D. Cells were injected subcutaneously into nude mice (10-11 mice per group). Intra-tumor injections of Ad5/35-tk viruses (1.5 × 10⁸ PFU) were made twice at the indicated time points followed by GCV injections (75 mg/kg) intraperitoneally on days 2–12. Tumor growth was measured by a caliper at the indicated time points. E. Cell proliferation of parental A549 and CD46-overexpressing A549 was measured by the MTT assay. Error bars represent SEM. Statistics: A-B, p<0.01 by 2-way ANOVA; D, p=0.005 by repeated-measures ANOVA; E, p=0.44 by 1-way ANOVA.

Figure 5. Immunohistochemical analysis of CD46 expression in patient colorectal cancers

Tumors were analyzed for the expression of CD46 by immunostaining. A, normal mucosa. B-C, well-differentiated tumors. D-F, poorly differentiated and highly invasive tumors. While CD46 was generally overexpressed in colorectal tumors compared to normal counterparts, some invasive cancers showed no expression of CD46. High-powered pictures are shown in inlets of selected areas.

Figure 6. CD46 expression indicates better survival of CRC patients

Overall survivability was demonstrated by Kaplan-Meier curve and measured by the log-rank test (p=0.031).