Abstract A008: An indocyanine green-based liquid biopsy test for circulating tumor cells for hepatoblastoma
Authors: Espinoza Andres F. , Patel Roma H. , Armbruster Bryan W. , Kureti Pavan , Govindu Saiabhiroop R. , Vasudevan Sanjeev A. , Woodfield Sarah E. 
Published: January 2023

Abstract
Introduction: Hepatoblastoma (HB) is the most common pediatric primary liver tumor and has the fastest rising incidence of all pediatric solid tumors. Tumor progression is monitored with MRI, chest CT, and serum levels of Alpha fetoprotein. Unfortunately, these assays involve exposing children to radiation and anesthesia and are not always accurate. The goal of this work is to develop and validate a liquid biopsy test for circulating tumor cells (CTCs) with a panel of HB-specific markers to provide a less invasive and more accurate way of assessing disease. Methods: We first explored accumulation of the far-red fluorescent dye indocyanine green (ICG) in widely available cell lines (HepG2, Huh-6, Hep3B, HepRG, 293T, SH-SY5Y, A549), our own HB patient-derived cell line (HB17), and primary fibroblasts. We assessed ICG accumulation with fluorescence microscopy and flow cytometry, including standard flow cytometry on a BD Symphony instrument and imaging flow cytometry on the Amnis ImageStream X MK II instrument. Based on this data, we then developed a new method for quantifying CTC burden from patient whole blood samples using HB-specific markers ICG, Glypican-3 (GPC3), and DAPI with fluorescence microscopy and flow cytometry readouts. We tested this panel with cell lines and non-cancer control blood samples, as well as blood samples from mice harboring intrahepatic patient-derived xenograft (PDX) tumors representing HB. Finally, we used this panel to analyze whole blood samples for CTC burden with a cohort of HB patients and correlated with patient characteristics and outcomes. Results: First, we showed that ICG accumulation is specific to liver cancer cells (HepG2, Huh-6, Hep3B, HB17), compared to non-malignant liver cells (HepRG), non-liver solid tumor cells (SH-SY5Y, A549), and non-malignant cells (293T, primary fibroblasts). In addition, we found that ICG+ liver tumor cells remain positive for at least 96 hours after uptake. We also showed that ICG accumulation can be used to identify HepG2 HB cells in a mixed population of cells down to a dilution of 1:100 (2.7% ICG+ in the HepG2/SH-SY5Y mix versus 1.9% ICG+ in the SH-SY5Y control). Flow cytometry with HepG2 and A549 cell lines with the ICG/GPC3/DAPI panel showed 94.4% of HepG2 cells were triple ICG+/GPC3+/DAPI+ while 0% of A549 cells were triple ICG+/GPC3+/DAPI+. With non-cancer control blood samples, we identified less than 1 ICG+ cell per ml of blood. We showed the presence of triple ICG+/GPC3+/DAPI+ cells in the blood of animals harboring PDX tumors. Most importantly, with a low-risk HB patient sample, we found 2.25 triple ICG+/GPC3+/DAPI+ cells per ml of blood; with a high-risk sample we counted 17.25 triple ICG+/GPC3+/DAPI+ cells per ml of blood. Conclusions: This work shows the translation from the use of ICG to identify HB lesions in patients during surgery to its role in a novel liquid biopsy test for CTCs, which has the real potential to improve the standard of care by offering a more accurate and less invasive means of monitoring disease.
Citation Format: Andres F. Espinoza, Roma H. Patel, Bryan W. Armbruster, Pavan Kureti, Saiabhiroop R. Govindu, Sanjeev A. Vasudevan, Sarah E. Woodfield. An indocyanine green-based liquid biopsy test for circulating tumor cells for hepatoblastoma [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A008.