There is a growing body of nuclear contrast agents that are repurposed for fluorescence guided surgery. New contrast agents are obtained by substituting the radioactive tag with, or adding a fluorescent cyanine to the molecular structure. This enables intra-operative fluorescent detection of cancerous tissue, leading to more complete tumor resection. However, these fluorescent cyanines can have a remarkable influence on pharmacokinetics and tumor uptake. Here we demonstrate the effect of cyanine-mediated dead cell binding of Ac-Lys0(IRDye800CW)-Tyr3-octreotate (800CW-TATE) and how this can be used as an advantage for the purpose of fluorescence guided surgery. We exposed cultured U2OS cells (alive or dead, with or without SSTR2-expression) and frozen human tumor tissue sections of NCI-H69 (SSTR2 positive) and CH-157MN (SSTR2 negative) to 800CW-TATE. We then injected 800CW-TATE into NCI-H69-tumor-bearing mice. Blocking experiment were included with DOTA0-Tyr3-octreotate (DOTA-TATE). Paraffin sections of the resected tumors were imaged for near infrared fluorescence and cell death staining was performed.
Binding of 800CW-TATE could be blocked with DOTA-TATE and was absent in SSTR2 negative cells. However, strong binding was observed to dead cells, which could not be blocked with DOTA-TATE and was also present in dead SSTR2 negative cells. No SSTR2-mediated binding was observed in frozen sections, possibly due to disruption of the cells in the process of sectioning the tissue before exposure to the contrast agent. DOTA-TATE blocking resulted in incomplete reduction of 61.5 ± 5.8% fluorescence uptake by the tumors in the mice. Paraffin sections revealed that fluorescence uptake persisted in necrotic regions upon blocking.
This study shows that labeling peptides with cyanines can result in dead cell binding. This does not hamper the ultimate purpose of fluorescence guided surgery, as necrotic tissue appears in most solid tumors. Hence, the necrosis binding can increase the overall tumor uptake. Moreover, necrotic tissue should be removed as much as possible: it cannot be salvaged, causes inflammation, and is tumorigenic. However, when performing binding experiments to cells with disrupted membrane integrity, which is routinely done with nuclear probes, this dead cell binding can resemble non-specific binding. This study will benefit development fluorescent contrast agents.