Objective: There is growing interest in reinforcing response to anticancer therapies by promoting immunogenic cell death. This mode of cell death induces surface exposure of calreticulin (CRT) in a manner that precedes apoptosis. In this study, we explored whether CRT-targeted optical imaging may have potential as a biomarker of immunogenic cell death.

Methods: We used a monoclonal antibody against human CRT (FMC 75) conjugated with a phycoerythrin label as a fluorescent imaging probe for CRT. Human CT-26 colon cancer cells were used for in vitro studies, and immunogenic cell death was induced by treatment with 25 M etoposide. Targeting of the probe to CRT exposed on the surface of etoposide-treated cells was verified by confocal microscopy and fluorescence-activated cell sorting (FACS) analysis. In vivo imaging was performed in tumor bearing Balb/C nude mice xenografted by subcutaneous injection of MDA-MB-468 human breast cancer cells. Tumors approximately 1 cm in diameter were treated with 100 μM etoposide intra-tumorally injected in 100 μl PBS. Phycoerythrin labeled anti-CRT antibody was injected into the tail vein 3 hr later. Optical imaging of fluorescent signals was performed on an IVIS® Spectrum system (Xenogen) at 4 and 21 hr post-injection using an excitation filter of 570 nm and emission filter of 620 nm.

Results: Confocal microscopy displayed specific binding of phycoerythrin anti-CRT antibodies to the surface of etoposide-treated CT-26 cancer cells. FACS analysis with the probe showed a right-shift of fluorescent signals in cells treated with etoposide. Hence, signal-positive cells were increased from 1.83 ± 0.1% to 5.81 ± 0.5% by 4 and 24 hr of treatment, respectively. In vivo optical imaging following intra-tumoral etoposide injection demonstrated clear tumor uptake of phycoerythrin anti-CRT antibody from 4 hr post-injection. Tumor contrast was substantially enhanced at 21 hr post-injection owing to reduction of background activity, which resulted in high tumor-to-muscle signal ratios.

Conclusion: This study provides the first evidence that targeted imaging of cell surface-exposed CRT may be useful for noninvasive monitoring of immunogenic cell death during anticancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B111.