A genetic strategy for combined screening and localized imaging of breast cancer

Abstract

Purpose: Improvements are needed for the early detection of breast cancer, as current imaging methods lack sensitivity to detect small tumors and assess their disease phenotype.

Procedures: To address this issue, the dual reporter adenoviral vector (Ad5/3-Id1-SEAP-Id1-mCherry) was produced with a cancer-specific Id1 promoter driving expression of a blood-based screening reporter (secreted embryonic alkaline phosphatase, SEAP) and a fluorescent imaging reporter (mCherry). This diagnostic system was assessed for its screening potential on breast cancer cell lines of various aggressive phenotypes. Reporter expression was measured and correlated with promoter level expression using Western blot. Adenovirus receptor expression was normalized against reporter expression with luciferase infectivity assays. Ad5/3-Id1-SEAP-Id1-mCherry infected MDA-MB-231 cells combined with uninfected cells were implanted into the mammary fat pad of athymic nude mice to recapitulate low-dose tumor delivery. Id1 driven SEAP expression and mCherry imaging were monitored to validate diagnostic sensitivity and efficacy.

Results: Infected breast cancer cell lines displayed SEAP levels in the media that were 10-fold above background by 2 days after infection. Ad5/3-Id1-SEAP-Id1-mCherry infected cells (multiplicity of infection = 10) implanted in athymic nude mice demonstrated a 14-fold increase in serum SEAP levels over baseline when as little as 2.5% of the tumor contained infected cells. This robust response was also found for the mCherry reporter, which was clearly visible in tumor xenografts on day 2 post implantation.

Conclusions: This diagnostic system that combines screening with imaging for early detection and monitoring of breast cancer can be easily extended to other reporters/modalities and cancer-targeting methods. Combining screening with imaging in a genetic, cancer-specific mechanism allows sensitive multi-modal detection and localization of breast cancer.

Fig 1

Studies with MCF7 and MDA-MB-231 for. (a) media SEAP over time, (b) media SEAP level as influenced by serum starvation (ss), (c) Id1 protein by Western blot analyses.. Data are means +/− SD.

Fig. 2

Comparison of breast cancer cell lines for (a) Id1 expression (via SEAP) and infectivity (via luciferase) and (b) fluorescence imaging of representative cell lines for Id1-specific mCherry. Data are means +/− SD.

Fig 3

Time course sensitivity study of MCF7 and MDA-MB-231 cells (100K and 350K) following infection with Ad5/3-Id1-SEAP-Id1-mCherry for (a) media SEAP, and (b)fluorescence (Representative fluorescent and inset bright field images are included). Images (20x) were collected using a 587nm excitation/600nm long pass filter and Nuance multi-spectral camera at 100ms exposure. Data are means +/− SD.

Fig 4

In vivo experiment with MDA-MB-231 tumor xenograft showing (a) SEAP levels (ng/ml of blood plasma, mean +/− SD), and imaging of mCherry on day 2 post implantation in tumor with (b) 5.0×10⁴ infected cells with matching excised tumor (right) removed 14 days post implantation. (c) 3.5×10⁵ infected cells with matching excised tumor (right) removed 14 days post implantation. Whole body images (0.63x) and excised tumor images (2.5x) were collected using a 587nm excitation/600nm long pass filter and Nuance multi-spectral camera at 4s exposure.