Investigating the mechanisms of indocyanine green (ICG) cellular uptake in sarcoma

IntroductionIndocyanine green (ICG) is a near infrared (NIR) dye which has been used clinically for over 50 years and has recently been utilised for fluorescence guided surgery in a number of cancer types, including sarcoma. ICG is taken up and retained by sarcoma tumours to a greater extent than normal tissue, demonstrating its potential to aid in visualisation of tumour margins. However, the mechanisms surrounding preferential ICG uptake in tumours are poorly understood. MethodsIn vitro ICG cellular uptake studies were performed across a panel of four sarcoma cell lines and one breast cancer cell line, 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. The spatial orientation of ICG was also assessed in a patient specimen. ResultsThe level of ICG cellular uptake was dependent on ICG concentration and incubation time. Cell line proliferation rate correlated significantly to ICG uptake within 30 minutes (Rs= 1, p<0.001), whilst retention of ICG after 24hrs did not (Rs= 0.3, p=0.624). From our data, the primary mechanism of ICG uptake in sarcoma cells is via clathrin mediated endocytosis. Following the resection of a grade 3 leiomyosarcoma, ICG signal was detectable macroscopically and on 3m sections, whilst being negative on the muscle control. ConclusionsThe use of ICG for tumour detection in sarcoma surgery may demonstrate higher utility in high grade tumours compared to low grade tumours, due to the observation of higher ICG uptake in more proliferative cell lines. It is likely that the enhanced permeability and retention (EPR) effect plays a significant role in the retention of ICG within tumours. Future work on the detection of ICG at the cellular level within human tissue sections is required, with the aid of purpose built NIR microscopes.