Protocol development of paired-agent fluorescent imaging to detect micrometastases in resected breast lymph nodes

Abstract

The presence of lymph node metastases played as a critical prognostic factor in breast cancer treatment and guiding the future adjuvant treatment. The possibility of missed micrometastases by conventional pathology was estimated around 20-60% cases has created a demand for the development of more accurate approaches. Here, a paired-agent imaging approach is presented that employs a control imaging agent to allow rapid, quantitative mapping of microscopic populations of tumor cells in lymph nodes to guide pathology sectioning. To test the feasibility of this approach to identify micrometastases, healthy rat and human lymph nodes were stained with targeted and control imaging agent solution to evaluate the potential for the agents to diffuse into and out of intact nodes. Erbitux, an EGFR specific antibody was labeled with IRDye-700DX(LICOR) as targeted agent and IRDye-800CW was labeled to rat IgG as control agent. Lymph nodes were stained for 60 min, followed by 30 min rinsing, and the uptake and washout of fluorescence were recorded. Subsequently, lymph nodes were frozen-sectioned and imaged under an 80- um resolution fluorescence imaging system (Pearl, LICOR) to confirm equivalence of spatial distribution of both agents in the entire node. Both imaging agents correlated well with each other(r=0.877) and the binding potential of targeted agent was found to be 0.08 ± 0.22 along the lymph node in the absence of binding. The results demonstrate this approach’s potential to enhance the sensitivity of lymph node pathology by detecting fewer than 1000 cell in a whole human lymph node.