Comparison of visible and near-infrared wavelength-excitable fluorescent dyes for molecular imaging of cancer

TLDR

Targeted fluorescent probes, especially stable organic fluorophores that can be repeatedly excited in vivo, offer high signal‑to‑noise imaging of disease, yet most small‑animal studies rely on the red‑excitable Cy5.5 dye. We compare the in‑vivo imaging performance of a near‑infrared dye versus a red‑excitable dye. EGF was conjugated to either Cy5.5 (660/710 nm) or IRDye® 800CW (785/830 nm) and injected intravenously (1 nmol dye) into mice bearing EGFr‑positive and –negative breast cancer xenografts, with or without pre‑administered anti‑EGFr antibody C225. Both conjugates targeted EGFr, but the IRDye® 800CW produced a markedly lower background and higher tumor‑to‑background ratio than Cy5.5, while immunohistochemistry revealed that EGF activates EGFr signaling, indicating possible adverse effects of using EGF as a targeting agent.

Abstract

Targeted fluorescent molecular imaging probes may provide an optimal means of detecting disease. Stable, organic fluorophores can be repeatedly excited in vivo by propagated light and consequentially can provide large signal-to-noise ratios (SNRs) for image detection of target tissues. In the literature, many small animal imaging studies are performed with a red excitable dye, Cy5.5, conjugated to the targeting component. We report the comparison of the in vivo fluorescent imaging performance of a near-IR (NIR) and a red-excitable dye. Epidermal growth factor (EGF) was conjugated with Cy5.5 [excitation/emission (ex/em), 660/710 nm] or IRDye® 800CW (ex/em: 785/830 nm) for imaging EGF receptor (EGFr) positive (MDA-MB-468) and/or negative (MDA-MB-435) human breast cancer cell lines in subcutaneous xenograft models. The conjugates were injected intravenously at 1-nmol-dye equivalent with and without anti-EGFr monoclonal antibody C225, preadministered 24 h prior as a competitive ligand to EGFr. Our images show that while both agents target EGFr, the EGF-IRDye® 800CW evidenced a significantly reduced background and enhanced the tumor-to-background ratio (TBR) compared to the EGF-Cy5.5. Immunohistochemistry shows that EGF causes activation of the EGFr signaling pathway, suggesting that prior to use as a targeting, diagnostic agent, potential deleterious effects should be considered.