Chan Corey David, Brookes Marcus J, Pringle Toni A, Pal Rahul, Tanwani Riya, Burt Alastair D, Knight James C, Kumar Anand Tn, Rankin Kenneth S
The North of England Bone and Soft Tissue Tumour Service, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, UK.
Newcastle University Centre for Cancer, Translational and Clinical Research Institute, Newcastle University, Newcastle upon Tyne, UK.
J Pathol. 2025 Sep 10. doi: 10.1002/path.6473.
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.
吲哚菁绿（ICG）是一种成熟的近红外染料，已在临床上使用了数十年。最近，它已被用于多种实体癌类型的荧光引导手术，包括肉瘤，目的是降低切缘阳性率。与正常组织相比，ICG在肿瘤内的摄取和保留增加，这使外科医生在通过近红外相机观察时能够获得视觉参考以辅助切除。然而，这一过程的机制尚不清楚。我们对一组具有不同增殖率和表型的肉瘤细胞系进行了体外ICG细胞摄取研究。使用荧光显微镜和流式细胞术研究了ICG浓度、孵育时间、网格蛋白介导的内吞作用抑制以及细胞系增殖率对ICG细胞摄取的影响。通过与溶酶体标记物共定位评估细胞内ICG的亚细胞定位。通过高分辨率组织成像评估荧光引导手术后患者肿瘤组织中ICG的空间分布，并使用荧光寿命成像进行量化。体外结果表明，细胞系增殖率与ICG摄取显著相关（斯皮尔曼等级相关系数 = 1.00，p < 0.001），孵育24小时后观察到最大ICG摄取。抑制网格蛋白介导的内吞作用可显著降低ICG细胞摄取（p = 0.0004），细胞内ICG在30分钟内与溶酶体标记物显著共定位（皮尔逊r = 0.8）。在对三种不同肉瘤亚型的肿瘤组织进行组织学分析时，在肉瘤细胞内观察到ICG，并在肿瘤微环境中的少量出血和坏死区域积聚。通过对ICG荧光寿命成像的量化，我们能够区分肿瘤组织中的肉瘤细胞与出血和坏死。将体外数据与患者组织分析相结合，我们提出ICG在肉瘤中的摄取和积聚是由一种协同机制驱动的，该机制涉及增强的通透性和滞留效应以及染料的活性肿瘤细胞内吞作用。© 2025作者。《病理学杂志》由约翰·威利父子有限公司代表大不列颠及爱尔兰病理学会出版。
