Lymphatic mapping and surgical lymphadenectomy guidance using fluorescently labeled mannose-receptor-targeting nanobodies

INTRODUCTION: Lymphadenectomy, the surgical removal of lymph nodes (LNs), is performed in oncology patients to control the lymphatic tissue for signs of cancer. Depending on the cancer type and stage, either a regional or radical LN dissection is performed. Intraoperative identification of LNs can be a complex and demanding procedure. We propose to use fluorescence molecular imaging to highlight all LNs within the surgical field to facilitate node picking. To accomplish LN staining, we fluorescently labeled a previously developed anti-mannose receptor (MR) nanobody (Nb) that yields significant uptake in the LNs.

METHODS: The nanobody MMR3.49 with high affinity for MR and a non-targeting control nanobody were labeled with Cy5 or IRDye800CW. One hour after intravenous injection of 2nmol of labeled compound in healthy mice (n=3 per group), axillary, inguinal and popliteal LNs were imaged in situ and ex vivo with the KIS650, Kaer Labs (for detection of Cy5 signal) or Fluobeam800, Fluoptics (for detection of IRDye800CW signal). The translational nature of the approach was explored by upscaling the experiment to healthy pigs (30-40kg; n=2 for Cy5, n=3 for IRDye800CW). Here, LN mapping in the cervical, abdominal, pelvic, and/or popliteal area was evaluated 90 minutes after intravenous injection of 73nmol of MMR3.49-Cy5/IRDye800CW. LNs and muscle were procured for further ex vivo assessment. Fluorescent signal and target-to-background ratios (TBRs) were quantified using ImageJ.

RESULTS: Both Cy5 and IRDye800CW-labeled anti-MR nanobodies enabled clear and specific in situ visualization of the LNs in mice, with ex vivo TBRs of 2.64±0.63 and 4.62±0.50 respectively. No uptake in LNs was observed for the control nanobodies (TBR 1.40±0.18 and 1.14±0.32) (Figure 1). Comparably, in pigs, MMR3.49-Cy5/IRDye800CW allowed distinct in vivo fluorescent staining of the LNs and facilitated localizing them in situ (Figure 2). Ex vivo TBRs were calculated at 2.61±1.11 for Cy5 and 3.57±0.93 for IRDye800CW labeled anti-MR nanobody.

CONCLUSIONS: Intravenous injection of fluorescently labeled anti-MR nanobodies enabled intraoperative localization of LNs within the whole surgical view. This could potentially increase the accuracy of both regional and radical nodal dissections. Early data suggests the imaging findings in mice are scalable to porcine models.