Investigating the mechanisms of indocyanine green tumour uptake in sarcoma cell lines and <i>ex vivo</i> human tissue

Indocyanine green (ICG) is a well-established near-infrared dye which has been used clinically for several decades. Recently, it has been utilised for fluorescence-guided surgery in a range of solid cancer types, including sarcoma, with the aim of reducing the positive margin rate. The increased uptake and retention of ICG within tumours, compared with normal tissue, gives surgeons a visual reference to aid resection when viewed through a near-infrared camera. However, the mechanisms of this process are poorly understood. 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 fluorescence-guided surgery was assessed by high-resolution tissue imaging and quantified using fluorescence lifetime imaging. In vitro results showed that the cell line proliferation rate correlated significantly with ICG uptake (Spearman's rank correlation coefficient = 1.00, p < 0.001), and maximum ICG uptake was observed after 24 h incubation. 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 of tumour tissue from three different sarcoma subtypes, ICG was observed within sarcoma cells as well as accumulating in paucicellular areas of haemorrhage and necrosis within the tumour microenvironment. Through quantification of fluorescence lifetime imaging of ICG, we were able to differentiate sarcoma cells from haemorrhage and necrosis within tumour tissue. Combining in vitro data with analysis of patient tissue, we propose that the uptake and accumulation of ICG in sarcomas is driven by a synergistic mechanism involving the enhanced permeability and retention effect combined with active tumour cell endocytosis of the dye. © 2025 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.