(P 22) MECHANISMS OF PREFERENTIAL TUMOUR UPTAKE OF INDOCYANINE GREEN IN SARCOMA

Objective: Indocyanine green (ICG) is a well-established near-infrared dye used for fluorescence guided surgery (FGS) in a range of solid cancer types, including sarcoma. The preferential uptake and retention of ICG within tumours allows surgeons to visualise tumour margins intraoperatively, helping to aid complete resection. The use of FGS technology for the resection of bone and soft tissue sarcomas is an evolving field, however the mechanisms of this process are poorly understood.

Methods: We performed in vitro ICG cellular uptake studies across a panel of sarcoma cell lines exhibiting varying proliferation rates and phenotypes. The effects of ICG concentration, incubation time, inhibition of clathrin-mediated endocytosis, and cell line proliferation rate on the cellular uptake of ICG were investigated using fluorescence microscopy and flow cytometry. Subcellular localisation of intracellular ICG was assessed via colocalization with a lysosomal marker. The spatial distribution of ICG in patient tumour tissue following FGS was assessed by high-resolution tissue imaging, immunofluorescent microscopy and quantified using fluorescence lifetime imaging.

Results: In vitro results showed that cell line proliferation rate correlated significantly with ICG uptake within 30 min (Rs = 1, p < 0.001). ICG cellular uptake was significantly reduced by inhibition of clathrin-mediated endocytosis (p = 0.0004), and intracellular ICG significantly colocalized with a lysosomal marker within 30 min (Pearson’s r = 0.8). On histological analysis, ICG was observed within tumour cells as well as accumulating in paucicellular areas of haemorrhage and necrosis. Quantification using fluorescence lifetime imaging showed that ICG was present in these two distinct locations within tumour tissue, independent of ICG concentration and tissue autofluorescence.

Conclusion: Combining in vitro data with analysis of patient tissue, we propose that the preferential uptake and accumulation of ICG in sarcomas is driven by the enhanced permeability and retention effect of the tumour bulk, combined with active tumour cell endocytosis. These findings will help optimise the clinical use of ICG for this application, and help guide the dose, timing of administration and post-operative tissue processing of sarcoma tissue.