Preclinical evaluation of transcriptional targeting strategies for carcinoma of the breast in a tissue slice model system

Abstract

Introduction: In view of the limited success of available treatment modalities for metastatic breast cancer, alternative and complementary strategies need to be developed. Adenoviral vector mediated strategies for breast cancer gene therapy and virotherapy are a promising novel therapeutic platform for the treatment of breast cancer. However, the promiscuous tropism of adenoviruses (Ads) is a major concern. Employing tissue specific promoters (TSPs) to restrict transgene expression or viral replication is an effective way to increase specificity towards tumor tissues and to reduce adverse effects in non-target tissues such as the liver. In this regard, candidate breast cancer TSPs include promoters of the genes for the epithelial glycoprotein 2 (EGP-2), cyclooxygenase-2 (Cox-2), alpha-chemokine SDF-1 receptor (stromal-cell-derived factor, CXCR4), secretory leukoprotease inhibitor (SLPI) and survivin.

Methods: We employed E1-deleted Ads that express the reporter gene luciferase under the control of the promoters of interest. We evaluated this class of vectors in various established breast cancer cell lines, primary breast cancer cells and finally in the most stringent preclinical available substrate system, constituted by precision cut tissue slices of human breast cancer and liver.

Results: Overall, the CXCR4 promoter exhibited the highest luciferase activity in breast cancer cell lines, primary breast cancer cells and breast cancer tissue slices. Importantly, the CXCR4 promoter displayed a very low activity in human primary fibroblasts and human liver tissue slices. Interestingly, gene expression profiles correlated with the promoter activities both in breast cancer cell lines and primary breast cancer cells.

Conclusion: These data suggest that the CXCR4 promoter has an ideal 'breast cancer-on/liver-off' profile, and could, therefore, be a powerful tool in Ad vector based gene therapy or virotherapy of the carcinoma of the breast.

Figure 1

Evaluation of candidate promoters for transcriptional targeting of breast cancer cell lines. Luciferase activities in breast cancer cell lines (MB-468, AU-565, GI-101 and MB-231). These cell lines were infected with AdCXCR4Luc, AdSurvivinLuc, AdCox2MLuc, AdSLPILuc, AdEGP-2Luc and AdCMVLuc, respectively, at a multiplicity of infection of 100. Gene expression was measured 24 h after infection and is presented for the candidate promoters as percentage of cytomegalovirus promoter activity in relative light units (RLU). Each bar presents the mean of three experiments (± standard deviation).

Figure 2

Analysis of candidate promoters for transcriptional targeting of adenovirus-mediated gene expression and correlation to the mRNA copy number of the corresponding genes in primary breast cancer cells. (a) Evaluation of candidate promoters for transcriptional targeting of primary breast cancer cells derived from patients. Primary breast cancer cells were isolated from eight patients and purified and infected with AdCXCR4Luc, AdSurvivinLuc, AdCox2MLuc, AdSLPILuc, AdEGP-2Luc or AdCMVLuc, at a multiplicity of infection of 100. Gene expression was measured 24 h after infection and is presented for the candidate promoters as percentage of cytomegalovirus promoter activity in relative light units (RLU). Each bar presents the mean of three experiments ± standard deviation. (b) Gene expression of candidate tumor-specific genes in breast cancer patient samples. Messenger RNA was extracted from human primary breast cancer samples from eight patients and reverse-transcribed into cDNA. Real-time PCR was performed to evaluate the expression of the genes encoding cyclooxygenase (Cox)-2, epithelial glycoprotein (EGP)-2, secretory leukoprotease inhibitor (SLPI), survivin and the α-chemokine SDF-1 receptor (CXCR4). The mRNA copy numbers are normalized by the glyceraldehyde-3-phosphate dehydrogenase copy number. Each bar presents the mean of three experiments ± standard deviation.

Figure 3

Comparison of gene expression profiles in breast cancer patient samples and normal human fibroblasts. Messenger RNA was extracted from (a) human primary breast cancer samples from eight patients and (b) human primary fibroblasts from three patients and reverse-transcribed into cDNA. Real-time PCR was performed to evaluate the expression of the genes encoding cyclooxygenase (Cox)-2, epithelial glycoprotein (EGP)-2, secretory leukoprotease inhibitor (SLPI), survivin and the α-chemokine SDF-1 receptor (CXCR4). The mRNA copy numbers are normalized by the glyceraldehyde-3-phosphate dehydrogenase copy number. Each bar presents the mean ± standard deviation. Asterisks indicate p < 0.05 for breast cancer versus normal fibroblasts.

Figure 4

Evaluation of candidate promoters for transcriptional targeting of primary breast cancer tissue slices. Human breast cancer tissue slices were obtained from primary breast cancer samples using the Krumdieck Tissue Slicer. Slices were infected with AdCXCR4Luc, AdSurvivinLuc, AdSLPILuc, AdEGP-2Luc, AdCox2MLucor AdCMVLuc at a multiplicity of infection of 500. Gene expression was measured 24 h after infection and is presented for the candidate promoters as percentage of cytomegalovirus promoter activity in relative light units (RLU). All data points are of triplicate slices; bars represent means ± standard deviation. Cox, cyclooxygenase; CXCR4, α-chemokine SDF-1 receptor; EGP, epithelial glycoprotein; SLPI, secretory leukoprotease inhibitor.

Figure 5

Analysis of candidate promoters for transcriptional targeting of adenovirus-mediated gene expression in normal breast tissue slices. Human normal breast tissue slices (from three patients undergoing mammoplasty) were infected with AdCXCR4Luc, AdSurvivinLuc, AdSLPILuc, AdEGP-2Luc, AdCox2MLuc or AdCMVLuc. Gene expression was measured 24 h after infection and is presented for the candidate promoters as percentage of cytomegalovirus promoter activity in relative light units (RLU). All data points are of triplicate slices; bars represent means ± standard deviation.

Figure 6

Visualization of luciferase expression pattern of AdCXCR4Luc and AdCMVLuc in normal breast tissue slices versus breast cancer tissue slices. The upper panels show the tissue morphology as revealed by hematoxylin-and-eosin staining of thin sections of (a) normal breast tissue slices and (b) breast tissue slices. Representative indirect immunfluorescent detection of luciferase expression is shown in the lower panels for (a) human normal breast tissue slices and (b) breast cancer tissue slices infected with AdCXCR4Luc and AdCMVLuc. AdCMVLuc infected (a) normal breast tissue slices (lower left panel) and (b) breast cancer tissue slices (lower left panel) show immunofluorescent detection of the luciferase protein (green fluorescence), whereas (a) AdCXCR4 infected normal breast tissue slices (lower right panel) do not display luciferase activity. In contrast, (b) AdCXCR4 infected breast cancer tissue slices reveal luciferase activity (lower right panel). DAPI (4'6-diamidino-2-phenylindole 2HCL) staining (blue) indicates nuclei. Magnification ×200.

Figure 7

Gene expression patterns in human liver tissue slices. Human liver tissue slices (from three donors) were infected with AdCXCR4Luc, AdSurvivinLuc, AdSLPILuc, AdEGP-2Luc, AdCox2MLuc or AdCMVLuc. A luciferase assay was performed after 24 h. Gene expression was measured 24 h after infection and is presented for the candidate promoters as percentage of cytomegalovirus promoter activity in relative light units (RLU). All data points are of triplicate slices; bars represent means ± standard deviation. Asterisks indicate p < 0.05 versus AdCMVLuc. Cox, cyclooxygenase; CXCR4, α-chemokine SDF-1 receptor; EGP, epithelial glycoprotein; SLPI, secretory leukoprotease inhibitor.