The utility of a tissue slice model system to determine breast cancer infectivity by oncolytic adenoviruses

Abstract

Background: Due to advances in viral design, oncolytic adenoviruses have emerged as a promising approach for treatment of breast cancer. Tumor tissue slices offer a stringent model system for preclinical evaluation of adenovirus therapies, since the slices retain a morphology and phenotype that more closely resembles the in vivo setting than cell line cultures, and this system has been shown to have utility in the evaluation of viral infectivity and replication. In this study, we evaluated the efficacy of viral infection and replication using a tropism-modified oncolytic adenovirus.

Methods: Breast tumor tissue slices were infected with a tropism-modified oncolytic adenovirus, and a wild-type adenovirus for comparison. Efficiency of infection was evaluated using fluorescent microscopy, as the viruses used have been modified to express red fluorescent protein. Replication of the viruses was evaluated with quantitative real-time polymerase chain reaction (PCR) to assay viral E4 genome copy number, a surrogate indicator for the number of virions. The breast tumor tissue slices were evaluated for the expression of CD46 expression by immunohistochemistry.

Results: Infection and replication of our tropism modified oncolytic virus has been observed in the breast cancer tissue slice model system and is comparative to wild-type virus. A qualitative increase in the number of cells showing red fluorescent protein (RFP) expression was observed correlating with increasing multiplicity of infection. Higher relative infectivity of the virus was observed in tumor tissue compared with normal breast tissue. Replication of the virus was demonstrated through increases in E4 copy number at 48 and 72 h after infection in human breast tumor slices.

Conclusions: We have shown that a tropism modified oncolytic adenovirus can infect and replicate in breast cancer tissue slices, which may be an important preclinical indicator for its therapeutic utility.

Figure 1. Fluorescence microscopy images of breast tumor tissue slices infected with Ad5/3-CXCR4-IX-mRFP1

Slices were infected with increasing multiplicities of infection at 5, 50, and 500 viral particles/cell and examined after 72 h.

Figure 2. Infectivity of breast cancer tissue slices compared with normal breast tissue slices obtained from the same patient

Analysis of fluorescent reporter expression in tumor tissue slices (Fig. 2A) slices and normal breast tissue slices (Fig. 2B) from the same patient infected with Ad5/3-CXCR4-E1A-IX-mRFP1 vector. Analysis of fluorescent reporter expression in tumor tissue slices (Fig. 2C) slices and normal breast tissue slices (Fig. 2D) from the same patient infected with Ad5-wt-IX-mRFP1 vector.

Figure 3. Immunohistochemical analysis of CD46 expression in breast tumor tissue slices

Representative staining pattern of sections using (A) an anti-CD46 monoclonal antibody or using (B) no primary antibody. An adjacent section was H&E stained for identification of histology.

Figure 4. Replication of oncolytic adenoviruses in breast tumor tissue slices

Viral copy number as an indicator of viral replication in breast tumor tissue slices from (A) Patient 1 and (B) Patient 2. Growth medium was collected at 24, 48, and 72 h after infection with Ad5/3-CXCR4-E1A-IX-mRFP1 or Ad5-wt-IX-mRFP1. Points represent the mean ± SD of three replicate samples.