B. W. Armbruster1, A. F. Espinoza1, R. H. Patel1, S. A. Vasudevan1, S. E. Woodfield1 1Baylor College Of Medicine, Pediatric Surgical Oncology Laboratory, Divisions Of Pediatric Surgery And Surgical Research, Michael E. DeBakey Department Of Surgery, Houston, TX, USA
Introduction: Use of indocyanine green (ICG), a fluorescent dye, has recently gained popularity in surgical oncology. It can be used in surgical treatments of various forms of liver cancer through its ability to be specifically retained in malignant liver cells. Given this novel approach to distinguishing tumor tissue in the operating room, we sought to investigate whether ICG can be used to unambiguously identify malignant liver cells in in vitro studies.
Methods: First, we incubated cells in culture with ICG for 1 hour, washed the cells, and then imaged them with a fluorescence microscope with an ICG filter cube at two time points, immediately after washing and 24 hours after washing. We then mixed ICG avid (malignant liver cells) and non-ICG avid (fibroblasts) cells and repeated the ICG incubation, washing, and imaging. Second, we used flow cytometry with an APC-Cy7 laser to show ICG signal in ICG avid and non-ICG avid (neuroblastoma) cells incubated with ICG for 1 hour, washed, and then analyzed.
Results: We used fluorescence microscopy with a specific ICG filter to show that ICG fluorescent signal is highly present in malignant liver cells while notably absent from non-malignant liver cells and non-liver cells. This retention pattern was further corroborated with a mixed population of cells where malignant liver cells selectively took up and retained ICG, but no appreciable amount of ICG was present in the fibroblast population. Similar results were found with flow cytometry experiments with malignant liver cells and neuroblastoma cells with the liver cells clearly accumulating ICG while the neuroblastoma cells were negative. Further, we mixed the malignant liver and neuroblastoma cells and showed that the positive ICG signal is proportional to the number of malignant liver cells in a mixed population.
Conclusion: Taken together, this data shows that malignant liver cells in vitro specifically uptake and accumulate ICG over extended periods of time, in clear contrast to non-malignant liver cells and non-liver cells. Thus, ICG can be used in the laboratory setting to unambiguously identify liver tumor cells. In addition, this work paves the way to future studies that focus on understanding why ICG accumulates specifically in liver cancer cells.