Biodistribution and safety assessment of bladder cancer specific recombinant oncolytic adenovirus in subcutaneous xenografts tumor model in nude mice

Abstract

Background: The previous works about safety evaluation for constructed bladder tissue specific adenovirus are poorly documented. Thus, we investigated the biodistribution and body toxicity of bladder specific oncolytic adenovirus Ad-PSCAE-UPII-E1A (APU-E1A) and Ad-PSCAE-UPII-E1A-AR (APU-E1A-AR), providing meaningful information prior to embarking on human clinical trials.

Materials and method: Conditionally replicate recombinant adenovirus (CRADs) APU-E1A, APU-EIA-AR were constructed with bladder tissue specific UroplakinII(UPII) promoter to induce the expression of Ad5E1A gene and E1A-AR fusing gene, and PSCAE was inserted at upstream of promoter to enhance the function of promoter. Based on the cytopathic and anti-tumor effect of bladder cancer, these CRADs were intratumorally injected into subcutaneous xenografts tumor in nude mice. We then determined the toxicity through general health and behavioral assessment, hepatic and hematological toxicity evaluation, macroscopic and microscopic postmortem analyses. The spread of the transgene E1A of adenovirus was detected with RT-PCR and Western blot. Virus replication and distribution were examined with APU-LUC administration and Luciferase Assay.

Results: General assessment and body weight of the animals did not reveal any alteration in general behavior. The hematological alterations of groups which were injected with 5x10(8) pfu or higher dose (5x10(9) pfu) of APU-E1A and APU-E1A-AR showed no difference in comparison with PBS group, and only slight increased transaminases in contrast to PBS group at 5x10(9) pfu of APU-E1A and APU-E1A-AR were observed. E1A transgene did not disseminate to organs outside of xenograft tumor. Virus replication was not detected in other organs beside tumor according to Luciferase Assay.

Conclusions: Our study showed that recombinant adenovirus APU-E1A-AR and APU-E1A appear safe with 5x10(7) pfu and 5x10(8) pfu intratumorally injection in mice, without any discernable effects on general health and behavior.

Fig. (1)

Schematic diagrams of constructed oncolytic adenovirus of APU-E1A, APU-E1A-AR and APU-LUC. APU-E1A is based on Ad5 wherein the Prostate Stem Cell Antigen Enhancer (PSCAE, 327bp) is inserted and endogenous E1a promoter has been replaced by the human UP II promoter. In APU-E1A-AR, the androgen receptor (AR) gene was inserted following E1A. APU-LUC, with LUC gene inserted after UPII, is used to detect the biodistribution of adenovirus.

Fig. (2)

Body weight, hepatic and hematological toxicity profile after increasing single doses of oncolytic adenovirus administration in immunocompetent mice. (A) Average weights of mice from injection to 56 day, immediately prior to euthanasia. (B) The average value for alanine aminotransferase (AST) and aspartate aminotransferase (ALT). (C) The lymphocyte percentage in white blood cell and (D) average value for platelet. Phosphate-buffered saline (PBS) administration was used in the control group. Mean values ± SD of 5–10 mice/group were depicted. * significant (P < 0.05) by One-way analysis of variance compared with the PBS group.

Fig. (3)

Macroscopic and microscopic postmortem analysis (×400 magnification). (A) Weights of organs at sacrifice (n = 5-10 for both groups). Error bars indicate the mean ± SEM. (B–E) H&E staining of the (B) tumor, (C) liver, (D) lung and (E) brain, from animals that were injected with 5×10⁹ pfu APU-E1A-AR. The cellular necrosis of tumor tissue are shown (circle), according to IPP analysis, the tumor necrosis was severe (68% of damage, score of 4). (F) The tumor necrosis area percentage (%) were calculated with software Image-Pro Plus (IPP) 5.1 (n = 5-10 for both groups). Error bars indicate the mean ± SEM. * P <0.05 comparing with PBS control.

Fig. (4)

Anti-E1A immunostaining (×400 magnification). Immunohischemistry for anti-E1A is shown on tumor (A-C), liver (D), lung (E), heart (F), brain (G), spleen (H) and kidney (I) from PBS-treated mice (A), APU-E1A-AR 5×10⁸ pfu treated mice (B) and APU-E1A-AR 5×10⁹ pfu treated mice (C-I).

Fig. (5)

Biodistribution of E1A transgene with qRT-PCR. Relative mean number of copies of E1A transgene (standardized to GAPDH) was qualified in triplicates using Rotor-Gene Real-Time Analysis Software 6.1. * P <0.05 comparing with PBS control.

Fig. (6)

(A) Western blot analysis of E1A protein. Tumor and other major organs (Liver, Lung, Brain) of high dose APU-E1A and APU-E1A-AR injected mice were detected in triplicates, and GAPDH was control. (B) Luciferase reporter assay for APU-LUC. Tumor and other major organs of 5×10⁸ pfu APU-LUC injected mice were detected in triplicates. * p<0.05 comparing with other organs.