Intraoperative micrometastases detection using paired-agent fluorescent imaging: trial-and-error protocol development

The status of lymph node is considered a critical prognostic factor for staging and guiding the future adjuvant treatment in many cancer types. The estimation of undetected micrometastases (0.2-2mm diameter) by conventional pathology was around 30-60% cases which has created a demand for the development of more fast and accurate approaches. In response, a paired-agent imaging approach is presented by employing a control imaging agent to allow rapid, quantitative mapping of microscopic cancer cells in lymph nodes to guide pathology sectioning. To identify the most feasible and effective protocol using this approach to detect micrometastases intraoperatively, swine cervical lymph nodes were used to evaluate the potential of different protocols for the agents to diffuse into and out of intact nodes. Aby-029, an anti-EGFR affibody molecule labeled with IRDye-800CW was used as targeted imaging agent, and the IRDye-700DX carboxylate was used as control agent. The time-course paired-agent fluorescence of whole lymph node were recorded to monitor the uptake and washout kinetics. Subsequently, lymph nodes were frozen-sectioned and imaged under an 85-um resolution fluorescence imaging system (Pearl, LICOR) to confirm equivalence of spatial distribution of both agents in the entire node. After much trial-and error, the intranodal infusion staining and rinsing protocol demonstrated promising results that both imaging agents shown strong correlation with each other in the absence of cancer cells (r=0.99, p<0.001). This methodology indicated the potential of using paired-agent imaging approach to allow rapid and sufficient detection of micrometastases in excised lymph nodes intraoperatively.