Peroperative lymph node mapping using fluorescently-labeled mannose receptor-specific nanobodies

Introduction

Lymphadenectomy, the surgical removal of lymph nodes (LNs), is performed in clinically node-negative patients who have a high chance of nodal metastases. For quality assurance, often a minimum number of LNs in the area of interest needs to be harvested. Intraoperative identification of LNs can be a demanding procedure. We propose to use fluorescence molecular imaging to highlight all LNs within the surgical field to facilitate node picking. To realize LN staining we fluorescently labeled a previously developed anti-mannose receptor (MR) nanobody (Nb) that yields significant uptake in LNs [1].

Methods

The nanobody MMR3.49 with high affinity for MR as well as a control nanobody were fluorescently-labeled with Cy5 or IRDye800CW. One hour after intravenous injection of 2 nmol of labeled compound in healthy mice (n=3 per group), axillary, inguinal and popliteal LNs were imaged in situ and ex vivo with the KIS (Kaer Labs) for Cy5 signals or Fluobeam800® (Fluoptics) for IRDye800CW signals. The translational nature of the approach was studied by upscaling the experiment to a healthy porcine model (n=1; 35 kg). Here, LN mapping in the pelvic area, following intravenous injection of 73 nmol of MMR3.49-Cy5, was evaluated 90 min after injection. LNs, muscle and fat were procured for further ex vivo assessment. Fluorescent signals and target-to-background ratios (TBRs) were quantified using ImageJ.

Results/Discussion

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 the pig, MMR3.49-Cy5 helped to realize distinct in vivo fluorescent staining of pelvic LNs (Figure 2).

Conclusions

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

References
1 Xavier, C, Blykers, A, Laoui, D, Bolli, E, Vaneyken, I, Bridoux, J, Baudhuin, H, Raes, G, Everaert, H, Movahedi, K, Van Ginderachter, JA, Devoogdt, N, Caveliers, V, Lahoutte, T, Keyaerts, M 2019, ‘Clinical Translation of [68Ga]Ga-NOTA-anti-MMR-sdAb for PET/CT Imaging of Protumorigenic Macrophages’, Mol Imaging Biol, 21, 898, Springer International Publishing