Activateable Imaging Probes Light Up Inside Cancer Cells

Always "on" Cy5.5 (a fluorescent molecule-conjugated to a control polyclonal antibody against immunoglobulin) and activatable indocyanine green ICG-conjugated to specific antibodies against HER2+/HER1+ can produce fluorescent signals as shown in red and green, respectively. Only the target tumors (HER2+ or HER1+) allow green fluorescense to be detected when each specific activatable ICG-conjugated antibody (green) is taken inside the cells and released from conjugation; however, all tumors as well as the entire mouse body produce detectable fluorescense when always "on" non-specific IgG-Cy5.5 (red) is injected. (Indocyanine green ICG-conjugated anti-HER2 trastuzumab targets only tumor with HER2+ markers. Indocyanine green ICG-conjugated anti-HER1 panitumumab targets only tumor with HER1+.)

Imaging can be used to help diagnose cancer as well as monitor tumor progression and response to treatment. The field of molecular imaging focuses on techniques capable of detecting specific molecular targets associated with cancer; the agents used for molecular imaging—often called probes—are multifunctional, with components that allow them to both interact with their molecular target and emit a detectable signal.

The CCR Molecular Imaging Program (MIP) designs, develops, and tests novel molecular imaging probes for the detection and characterization of cancer. Mikako Ogawa, Ph.D., a postdoctoral fellow, and other MIP researchers recently developed a new type of probe that is activated only after entering a target cell. The probes are described in a recently published Cancer Research article.

Ogawa and colleagues created the new probes by combining a fluorescent molecule—or fluorophore—called indocyanine green (ICG) with monoclonal antibodies that recognize specific tumor-associated molecules. ICG is a near-infrared fluorophore, which are easier to detect through layers of tissue and produce less background than molecules that emit detectable light. Despite these advantages, use of ICG for molecular imaging has been limited because attachment of a targeting molecule dramatically reduces the fluorescence of ICG, making it difficult or impossible to detect.

Undeterred by the challenges of working with ICG, the MIP researchers decided to harness the fluorophore's characteristics to create a new type of imaging probe. ICG was linked to three different monoclonal antibodies. The target molecules of the antibodies—interleukin 2 receptor, EGFR1, and HER2—are often overexpressed on the surfaces of certain types of cancer cells.

As expected, once linked to the antibodies, ICG fluorescence was undetectable. Experiments in cultured cells initially echoed this observation: measurements taken shortly after the probes were applied to target-expressing cells revealed virtually no signal. However, after several hours, the glow of ICG was detected in cells expressing the target molecule. The researchers hypothesize that the probe is internalized and broken down; this degradation results in separation of ICG from the antibody, allowing the fluorophore to once again emit light.

The activateable probes were also tested in mice, each of which harbored target-expressing tumors and control tumors lacking the target. The animals were injected with the appropriate probe and imaged periodically over several days. ICG gradually began to glow within the cells of tumors expressing the molecular targets while tumors lacking the markers remained dim. These experiments verify the ability of activateable antibody-ICG probes to detect and monitor tumors with specific molecular markers in a physiological environment.

Development of these novel probes paves the way for use of a promising fluorophore—ICG—for molecular imaging of cancers that express a wide array of molecular markers. Before they can be used in the clinic, additional research is needed to confirm the safety and effectiveness of the probes, but ICG and all of the antibodies used in this study are approved by the FDA, which suggests the outlook for translation of these agents to the clinic is promising.

Summary Posted: Sun, 02/01/2009


Cancer Res. 2009 Feb 15;69(4):1268-72. Epub 2009 Jan 27 PubMed Link